Bacillus megaterium bioactive compositions and metabolites

ABSTRACT

Provided are bioactive compositions and metabolites derived from  Bacillus  and particularly  Bacillus megaterium  cultures responsible for controlling pests as well as their methods of use for controlling pests. Further provided are pesticidal  Bacillus megaterium  strains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/683,154, filed Aug. 14, 2012; the disclosure of whichis incorporated by reference in its entirety for all purposes.

TECHNICAL FIELD

The present disclosure is in the field of bioactive compositions havingpesticidal activity and methods for their use in controlling plantpests. In particular, such compositions comprise a Bacillus strainand/or its metabolites, more particularly strains of Bacillus megateriumand their metabolites.

BACKGROUND

Natural products are substances produced by microbes, plants, and otherorganisms. Microbial natural products offer an abundant source ofchemical diversity, and there is a long history of utilizing naturalproducts for pharmaceutical purposes. Despite the emphasis on naturalproducts for human therapeutics, where more than 50% are derived fromnatural products, only 11% of pesticides are derived from naturalsources. Nevertheless, natural product pesticides have a potential toplay an important role in controlling pests in both conventional andorganic farms. Secondary metabolites produced by microbes (bacteria,actinomycetes and fungi) provide novel chemical compounds which can beused either alone or in combination with known compounds to effectivelycontrol insect pests and to reduce the risk for resistance development.There are several well-known examples of microbial natural products thatare successful as agricultural insecticides (Thompson et al., 2000;Arena et al., 1995; Krieg et al. 1983).

The development of a microbial pesticide starts with the isolation of amicrobe in a pure culture. It then proceeds with efficacy and spectrumscreening using in vitro, in vivo or pilot scale trials in a greenhouseand in the field. At the same time, active compounds produced by themicrobe are isolated and identified. For the commercialization of amicrobial pesticide, the microbe has to be economically produced byfermentation at an industrial scale, and formulated with a biocompatiblecarrier and approved additives to increase efficacy and to maximize theease of application.

Uses of Bacillus Megaterium and Products Produced Therefrom

Bacillus megaterium is a Gram-positive bacterium that grows in simplemedia and on more than 62 out of 95 carbon sources, such astricarboxylic acid cycle intermediates (e.g., formate and acetate), andforms spores mainly under aerobic conditions (see, for example, Vary,2007). It has been found in a variety of habitats, such as soil,seawater, sediment, rice paddies, honey, fish, and dried food.

Bacillus megaterium has been found to have a number of different uses.Specifically, it produces a variety of industrial enzymes such aspenicillin acylase, various amylases, and glucose dehydrogenase(reviewed in, Vary, 2007). Additionally, a fermentation of B. megateriumATCC 19213 grown to stationary phase was found to produceN-Deoxyschizokinen, a siderophore, which was identified as4-[(3(acetylhydroxyamino) propyl) amino]-2-[2-[(3-(acetylamino) propyl)amino]-2-oxoethyl]-2-hydroxy-4-oxo-butanoic acid (Hu X and Boyer G. L,1995). Schizokinen, a citrate-containing dihydroxamate, a siderophorehas been produced by B. megaterium and Anabaena sp (Plowman J. E. et al1984). The involvement of the citrate α-hydroxycarboxylate moiety iniron chelation was investigated by comparing the iron binding behaviorof schizokinen with that of acetylschizokinen, a derivative in which thecitrate hydroxyl group was modified by acetylation.

Another set of uses for products derived from Bacillus megaterium hasbeen medicinal uses. BMG 59-R2, a peptide antibiotic, has been reportedfrom B. megaterium (FERM-p 6177). The compound also inhibits alkalinephosphatase and tumour growth (Japan. Pat., 83 164 561. (1983)).Fermentation culture of B. megaterium in the presence of ansatrieninproduces T23V and T23VI (Damberg, M. et al 1982). These compounds belongto the class of macrolides antibiotics, which also exhibit antitumoractivity. A nucleoside named oxetanocin was isolated from B. megateriumNK84-0218 and the structure was determined to be9-[(2R,3R,4S)-3,4-bis(hydroxymethyl)-2-oxetanyl]adenine by X-raycrystallographic analysis (Shimada N. et al., 1986). Oxetanocin showedactivity against herpes simplex virus-II (DNA virus) at 5.8 pg/well (50%inhibition of cytopathic effect), while the cytotoxicity against Verocells was 132.6 μg/well (50% inhibition of cell growth). Later, thederivatives of oxetanocin such as oxetanocins H, X, G and2-aminooxetanocin A (Shimada N. et al., 1987) are isolated from the samestrain which showed antiviral activities against herpes simplex virustype-II (HSV-II) and antiviral activities against human immunodeficiencyvirus. B. megaterium IFO 12108 (Nakahama, K. et al., 1981) was used forthe microbial transformation of anamtiocin, an antitumor antibioticproduced by Nocardia sp. C-15003 (N-1). Ansamitocin P-3 was convertedinto 15-hydroxyansamitocin P-3 (PHO-3), and 15-epi-15-hydroxyansamitocinP-3 (epi-PHO-3), by using B. megaterium (Izawa M. et al., 1981). Themicrobial conversion product of P-3, has greater antitumor activitiesagainst P 388 and L 1210 than the substrate P-3.

Various isolates of Bacillus megaterium have been used as insecticides,bactericides, fungicides and nematicides (see, for example, Aksoy, H. M.2008; U.S. Pat. Nos. 6,599,503, 7,906,131, 7,935,360). Some of these B.megaterium isolates have been used in combination with other bacteria totreat sludge and wastes such as Artemisia annua residue, flue dust, branpowder, feces of livestock and poultry, peat, and crop straw (see, forexample, U.S. Pat. No. 7,279,104).

SUMMARY

Provided is a B. megaterium having the following characteristics:

(A) pesticidal activity;

(B) produces a pesticidal compound having the following properties: (1)has a molecular weight of about 140-185 as determined by LiquidChromatography/Mass Spectroscopy (LC/MS); (2) has ¹H NMR values of δ7.28 (2H), 7.19 (2H), 7.17, 2.67, 2.31, 1.92 and has ¹³C NMR values of177.5, 142.9, 129.5, 129.5, 129.4, 129.4, 126.9, 36.1, 34.4, 28.1 (3)has a High Pressure Liquid Chromatography (HPLC) retention time of about8-18 minutes, on a reversed phase C-18 HPLC (Phenomenex, Luna 5μ C18(2)100 A, 100×4.60 mm) column using a water:acetonitrile (CH₃CN) gradientsolvent system (0-20 min; 90-0% aqueous CH₃CN, 20-24 min; 100% CH₃CN,24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90% aqueous CH₃CN) at 0.5mL/min flow rate and UV detection at 210 nm.;

(C) is non-pathogenic to vertebrate animals; and

(D) is susceptible to tetracycline, erythromycin, streptomycin,penicillin, ampicillin, Oxytetracycline, Chloramphenicol, Ciprofloxacin,Gentamicin, Piperacillin, Imipenem and Sulphamethoxazole-Trimethoprim.

Also provided is a supernatant, cell fraction, filtrate, extract,compound or metabolite derived from a culture of B. megaterium.

Furthermore, the Bacillus megaterium sp. may have a 16S rRNA genesequence comprising at least one of:

(A) a forward sequence having at least 99% identity and moreparticularly at least 99.5% identity to the sequence set forth in SEQ IDNO:1, a reverse sequence having at least 99% identity to the sequenceset forth in SEQ ID NO:2 and at least 99% identity to the consensussequence set forth in SEQ ID NO: 3;

(B) a forward sequence having at least 99% identity and moreparticularly at least 99.5% identity to the sequence set forth in SEQ IDNO:4, a reverse sequence having at least 99% identity to the sequenceset forth in SEQ ID NO:5 and at least 99% identity to the consensussequence set forth in SEQ ID NO: 6;

(C) a forward sequence having at least 99% identity and moreparticularly at least 99.5% identity to the sequence set forth in SEQ IDNO:7, a reverse sequence having at least 99% identity to the sequenceset forth in SEQ ID NO:8 and at least 99% identity to the consensussequence set forth in SEQ ID NO: 9.

Additionally, the Bacillus megaterium sp. may have a recA gene sequencecomprising at least one of:

(A) a forward sequence having at least 99% identity and moreparticularly at least 99.5% identity to the sequence set forth in SEQ IDNO:14, a reverse sequence having at least 99% identity and moreparticularly at least 99.5% identity to the sequence set forth in SEQ IDNO:15 and at least 99% identity and more particularly at least 99.5%identity to the consensus sequence set forth in SEQ ID NO: 16;

(B) a forward sequence having at least 99% identity and moreparticularly at least 99.5% identity to the sequence set forth in SEQ IDNO:17, a reverse sequence having at least 99% identity and moreparticularly at least 99.5% identity to the sequence set forth in SEQ IDNO:18 and at least 99% identity and more particularly at least 99.5%identity to the consensus sequence set forth in SEQ ID NO: 19;

(C) a forward sequence having at least 99% identity and moreparticularly at least 99.5% identity to the sequence set forth in SEQ IDNO:20, a reverse sequence having at least 99% identity to the sequenceset forth in SEQ ID NO:21 and at least 99% identity and moreparticularly at least 99.5% identity to the consensus sequence set forthin SEQ ID NO: 22.

In particular, the Bacillus is a B. megaterium strain having theidentifying characteristics of B. megaterium strain H491 (NRRL AccessionNo. B-50769), Bacillus megaterium strain M018 (NRRL Accession No.B-50770) and Bacillus megaterium strain J142 (NRRL Accession No.B-50771), or a strain derived from one of said strains (e.g., a mutantstrain). Therefore, in a related aspect, said B. megaterium is provided.Also provided is a substantially pure culture, or whole cell brothcomprising said microorganism or cell fraction, supernatant, filtrate,extract, compound or metabolite derived therefrom.

The compound used in the methods and compositions and combinations maybe a compound that

(A) has pesticidal activity;

(B) has a molecular weight of about 140-185 as determined by LiquidChromatography/Mass Spectroscopy (LC/MS) and

(C) has a High Pressure Liquid Chromatography (HPLC) retention time ofabout 8-18 minutes on a reversed phase C-18 HPLC column using awater:acetonitrile (CH₃CN) gradient solvent system (0-20 min; 90-0%aqueous CH₃CN, 20-24 min; 100% CH₃CN, 24-27 min; 0-90% aqueous CH₃CN,27-30 min; 90% aqueous CH₃CN) at 0.5 mL/min flow rate and UV detectionat 210 nm and

(D) is optionally obtainable from a B. megaterium. The compound in oneembodiment may be a polyketide.

In a particular embodiment, the compound may be derived from B.megaterium and has a monosubstituted aromatic polyketide structurecomprising at least one acid moiety, at least one 6 membered aromaticring and at least three methylene group; a molecular weight from 140 toabout 185 in the core structure; at least 8 carbons and at least 2oxygens.

In one specific embodiment, the compound

(A) is obtainable from a B. megaterium;

(B) is toxic to a pest;

(C) has a molecular weight of about 140-185 and more particularly, 164as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS);

(D) has ¹H NMR values of δ 7.28 (2H), 7.19 (2H), 7.17, 2.67, 2.31, 1.92and has ¹³C NMR values of 177.5, 142.9, 129.5, 129.5, 129.4, 129.4,126.9, 36.1, 34.4, 28.1

(E) has an High Pressure Liquid Chromatography (HPLC) retention time ofabout 8-18 minutes, more specifically about 12 minutes and even morespecifically about 12.16 min on a reversed phase C-18 HPLC (Phenomenex,Luna 5μ C18(2) 100 A, 100×4.60 mm) column using a water:acetonitrile(CH₃CN) gradient solvent system (0-20 min; 90-0% aqueous CH₃CN, 20-24min; 100% CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90% aqueousCH₃CN) at 0.5 mL/min flow rate and UV detection at 210 nm.

In a more particular embodiment, provided are compounds including butnot limited to:

(A) a compound having the structure ##STR001##

or a pesticidally acceptable salt or stereoisomers thereof, wherein n is0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; X is O, NH, NR or S; R is lower chainalkyl containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl moieties, aryl orarylalkyl moiety, substituted lower alkyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl,substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl,substituted thioalkyl, hydroxy, halogen, amino, amido or carboxy; R₁,R₂, R₃, R₄, R₅, R₆ are each independently H, hydroxyl, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, cycloalkyl, substitutedcycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substitutedthioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl,oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl;

(B) a compound having the structure ##STR001a##

or a pesticidally acceptable salt or stereoisomers thereof, wherein X isO, NH, NR or S; R is lower chain alkyl containing 1, 2, 3, 4, 5, 6, 7, 8or 9 alkyl moieties, aryl or arylalkyl moiety, substituted lower alkyl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amidoor carboxy; n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R₁, R₂, R₃, R₄ are eachindependently H, hydroxyl, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl;

(C) a compound having the structure ##STR001b##

or a pesticidally acceptable salt or stereoisomers thereof, wherein n is0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R₁, R₂, R₃, R₄, R₅ are eachindependently H, hydroxyl, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl;

In a more particular embodiment, the compound is the aromaticpolyketide, 4-phenylbutanoic acid (1).

These compounds may be obtained by

(A) culturing a B. megaterium in a culture medium, to obtain a B.megaterium whole-cell broth, under conditions sufficient to produce saidcompound(s), and

(B) isolating said compound(s) produced in (A) from the whole cell brothof (A).

In particular, the compound in step (B) may be isolated by (i) applyingthe whole cell broth to at least one of an ion exchange column, a sizeexclusion column or a reversed phase HPLC column to obtain columnfractions; (ii) assaying the column fractions for pesticidal activityand (iii) concentrating column fractions of (ii) to obtain the isolatedcompound. Alternatively, said compound(s) can be produced by chemicalsynthesis and the product(s) can be used either as a pure compound or asa crude product.

In additional embodiments, compositions and methods for modulating plantgrowth and in particular, promoting plant growth and/or modulating seedgermination and particularly promoting seed germination are provided.For example, culture medium obtained after growth of the B. megateriumstrains disclosed herein, also denoted whole cell broth (WCB), can beapplied to plants, seeds and/or their growth substrate (e.g., soil) topromote the growth of the seeds and plants or germination of the seedsof the plant. Alternatively, plants, seeds and/or their growth substrate(e.g., soil) can be inoculated with any one or combination of the B.megaterium strains disclosed herein for the purpose of promoting growthof the plant. In further embodiments, a substantially pure culture orcell broth comprising B. megaterium or a supernatant, cell fraction,filtrate, extract, compound and/or metabolite derived therefrom can beused in methods for plant growth promotion or seed germination.

In additional embodiments, any of the compounds disclosed herein can beused in methods for promoting the growth of plants; for example, acompound that:

(a) has a molecular weight of about 140-185 and more particularly, 164as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS);

(b) has ¹H NMR values of δ 7.28 (2H), 7.19 (2H), 7.17, 2.67, 2.31, 1.92and has ¹³C NMR values of 177.5, 142.9, 129.5, 129.5, 129.4, 129.4,126.9, 36.1, 34.4, 28.1; and

(c) has an High Pressure Liquid Chromatography (HPLC) retention time ofabout 8-18 minutes, more specifically about 12 minutes and even morespecifically about 12.16 min on a reversed phase C-18 HPLC (Phenomenex,Luna 5/μ C18(2) 100 A, 100×4.60 mm) column using a water:acetonitrile(CH₃CN) gradient solvent system (0-20 min; 90-0% aqueous CH₃CN, 20-24min; 100% CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90% aqueousCH₃CN) at 0.5 mL/min flow rate and UV detection at 210 nm.

Additional compounds useful in the disclosed methods and compositionsfor promoting plant growth include those identified above as STR001,STR001a STR001b and 4-phenylbutanoic acid (Compound 1).

Further provided is a pesticidal combination which may be synergistic toat least one pest comprising as active components: (a) a substantiallypure culture or cell broth comprising B. megaterium or a supernatant,cell fraction, filtrate, extract, compound and/or metabolite derivedtherefrom and (b) another pesticidal substance, wherein (a) and (b) maybe present in synergistic amounts. The pest, in a particular embodiment,may be an insect pest, but may also include, but is not limited to, anematode, plant fungus, plant virus and plant bacteria and weeds.Further, the combination may be a composition. The pesticidal substancemay be (a) derived from a microorganism; (b) a natural product and/or(c) a chemical pesticide and in particular a chemical nematicide.

In particular, the combination may comprise a whole microorganism,supernatant, filtrate and/or extract of B. megaterium and a pesticidalsubstance derived from a microorganism including but not limited toBacillus sp. (e.g., Bacillus thuringiensis or Bacillus thuringiensiskurstaki) and spinosad. Alternatively, the combination may comprise asupernatant, filtrate and/or extract of B. megaterium and a pesticidalsubstance derived from a natural product such as pyrethrum.Alternatively, the combination may comprise a supernatant, filtrateand/or extract of B. megaterium and a pesticidal substance which is achemical pesticide, particularly, an insecticide, where the insecticideincludes but is not limited to pyrethrins, spirotetramet andorganochlorines.

In a related aspect, provided herein is a method for modulating, inparticular, synergistically modulating infestation of at least one pestor pest species in or around a plant comprising applying to a plantand/or seeds thereof and/or substrate for growing said plant thecombinations set forth above with an amount of the combination effectiveto modulate infestation of said pest or pest species. Also providedherein are isolated compounds obtainable or derived from B. megateriumor alternatively, organisms capable of producing these compounds thatcan be used to control various pests, and/or also particularly,nematicidal pests.

Further provided is the use of

(a) a compound which:

-   -   (1) has a molecular weight of about 140-185 as determined by        Liquid Chromatography/Mass Spectroscopy (LC/MS);    -   (2) has ¹H NMR values of δ 7.28 (2H), 7.19 (2H), 7.17, 2.67,        2.31, 1.92 and has ¹³C NMR values of 177.5, 142.9, 129.5, 129.5,        129.4, 129.4, 126.9, 36.1, 34.4, 28.1;    -   (3) has an High Pressure Liquid Chromatography (HPLC) retention        time of about 8-18 minutes, on a reversed phase C-18 HPLC        (Phenomenex, Luna 5μ C18(2) 100 A, 100×4.60 mm) column using a        water:acetonitrile (CH₃CN) with a gradient solvent system (0-20        min; 90-0% aqueous CH₃CN, 20-24 min; 100% CH₃CN, 24-27 min;        0-90% aqueous CH₃CN, 27-30 min; 90% aqueous CH₃CN) at 0.5 mL/min        flow rate and UV detection of 210 nm.;    -   (4) has pesticidal properties; and

(b) optionally another substance, wherein said substance is a pesticide,effective to modulate pest infestation;

to formulate a pesticidal composition and/or modulate the infestation ofone or more pests.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of purification scheme forobtaining the compounds of the invention from culture broth.

FIG. 2 depicts the (−) ESI-LCMS data for 4-Phenylbutanoic acid (1).

FIG. 3 depicts ¹H NMR for 4-Phenylbutanoic acid (1) in CD₃OD-d₄ at 600MHz.

FIG. 4 depicts ¹³C NMR for 4-Phenylbutanoic acid (1) in CD₃OD-d₄ at 150MHz.

FIG. 5 depicts the average nematicidal activity of each batch for eachfermentation numbered 1-19 in chronological order.

FIG. 6 shows the efficacy of H491 supernatant in the control of M. haplaat different dilution rates.

FIG. 7 shows the results of two different 96-well plate extractbioassays of H491 fractions 1-6 and crude extract. Three controls areincluded in each trial; 1 positive (1% Avid) & 2 negative (DMSO &water). Trial 1 (T1) was carried out using M. hapla nematodes and thefractions were dissolved in 100% DMSO; and trial 2 (T2) was carried outusing M. incognita nematodes and the fractions were dissolved in DMSO.The % immotility is graphed in its corrected form where the reportedimmotility of DMSO blank is subtracted from each of samples tested.

FIG. 8 shows the results of three different 96-well plate extractbioassays of H491 purified peaks H1-H38. Three controls are included ineach trial; a positive control (1% Avid) and 2 negative controls (DMSO &water). Column load (Initial) and flow-through (Wash) were also assayed.Trial 1 (T1) was carried out with M. hapla nematodes, trials 2 and 3 (T2and T3) were carried out with M. incognita nematodes. Results for eachsample (fraction or control) are presented as a set of three bars: theleftmost bar show results for Trial 1 (M. hapla), the center bar showsresults for Trial 2 (M. incognita) and the rightmost bar shows resultsfor Trial 3 (M. incognita).

FIG. 9 shows the effects of H491 supernatant on M. incognita in cucumberin a water-agar-assay.

FIG. 10 shows nematicidal dose-response for H491 WCB in cucumber plantstested in a mini tube in planta assay. H491 WCB (identified as MBI303 inthe Figure) was used at full strength (1×) and at 0.7× and 0.5×dilutions. Fresh top weight (FTW, left-most bar in each set of three),fresh root weight (FRW, center bar in each group of three) and gallindex (right-most bar in each set of three) were measured for the threeconcentrations of H491 WCB and for plants treated with water (negativecontrol) and with Avid (Positive control).

FIG. 11 shows results of motility assays (expressed as percentage ofimmotile nematodes) conducted with WCB obtained from four differentfermentation batches of B. megaterium H491. Also shown are results fornematodes treated with water (negative control) and Avid (positivecontrol).

FIG. 12 shows the effect of 4-phenylbutanoic acid (Compound I), atdifferent concentrations (indicated on the abscissa) on juvenilenematode (J2) motility. Results for negative controls (DMSO and water)and a positive control (Avid) are also shown.

FIG. 13 shows the effects of three different strains of B. megaterium(H491, M018 and J142) on M. incognita J2 motility. Avid was used as apositive control, and water as a negative control.

DETAILED DESCRIPTION

While the compositions and methods heretofore are susceptible to variousmodifications and alternative forms, exemplary embodiments will hereinbe described in detail. It should be understood, however, that there isno intent to limit the invention to the particular forms disclosed, buton the contrary, the intention is to cover all modifications,equivalents, and alternatives falling within the spirit and scope of theinvention as defined by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is included therein. Smaller ranges are also included. Theupper and lower limits of these smaller ranges are also includedtherein, subject to any specifically excluded limit in the stated range.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

As defined herein, “derived from” means directly isolated or obtainedfrom a particular source or alternatively having identifyingcharacteristics of a substance or organism isolated or obtained from aparticular source. In the event that the “source” is an organism,“derived from” means that it may be isolated or obtained from theorganism itself or from the medium used to culture or grow saidorganism.

As defined herein, “whole broth culture” refers to a liquid culturecontaining both cells and media. If bacteria are grown on a plate thecells can be harvested in water or other liquid, whole culture.

The term “supernatant” refers to the liquid remaining when cells thatare grown in broth or harvested in another liquid from an agar plate areremoved by centrifugation, filtration, sedimentation, or other meanswell known in the art.

As defined herein, “filtrate” refers to liquid from a whole brothculture that has passed through a membrane.

As defined herein, “extract” refers to liquid substance removed fromcells by a solvent (water, detergent, buffer) and separated from thecells by centrifugation, filtration or other method.

As defined herein, “metabolite” refers to a compound, substance orbyproduct of a fermentation of a microorganism, or supernatant,filtrate, or extract obtained from a microorganism that has pesticidaland particularly, nematicidal activity.

As defined herein, an “isolated compound” is essentially free of othercompounds or substances, e.g., at least about 20% pure, preferably atleast about 40% pure, more preferably about 60% pure, even morepreferably about 80% pure, most preferably about 90% pure, and even mostpreferably about 95% pure, as determined by analytical methods,including but not limited to chromatographic methods and electrophoreticmethods.

A “carrier” as defined herein is an inert, organic or inorganicmaterial, with which the active ingredient is mixed or formulated tofacilitate its application to plant or other object to be treated, orits storage, transport and/or handling.

The term “modulate” as defined herein is used to mean to alter theamount of pest infestation or rate of spread of pest infestation.

The term “pest infestation” as defined herein, is the presence of a pestin an amount that causes a harmful effect including a disease orinfection in a host population or emergence of an undesired weed in agrowth system.

A “pesticide” as defined herein, is a substance derived from abiological product or chemical substance that increase mortality orinhibits the growth rate of plant pests and includes but is not limitedto nematicides, insecticides, plant fungicides, plant bactericides, andplant viricides.

As used herein, the term “alkyl” refers to a monovalent straight orbranched chain hydrocarbon group having from one to about 12 carbonatoms, including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-hexyl, and the like.

As used herein, “substituted alkyl” refers to alkyl groups furtherbearing one or more substituents selected from hydroxy, alkoxy,mercapto, cycloalkyl, substituted cycloalkyl, heterocyclic, substitutedheterocyclic, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, aryloxy, substituted aryloxy, halogen, cyano, nitro, amino,amido, —C(O)H, acyl, oxyacyl, carboxyl, sulfonyl, sulfonamide, sulfuryl,and the like.

As used herein, “alkenyl” refers to straight or branched chainhydrocarbyl groups having one or more carbon-carbon double bonds, andhaving in the range of about 2 up to 12 carbon atoms, and “substitutedalkenyl” refers to alkenyl groups further bearing one or moresubstituents as set forth above.

As used herein, “alkynyl” refers to straight or branched chainhydrocarbyl groups having at least one carbon-carbon triple bond, andhaving in the range of about 2 up to 12 carbon atoms, and “substitutedalkynyl” refers to alkynyl groups further bearing one or moresubstituents as set forth above.

As used herein, “aryl” refers to aromatic groups having in the range of6 up to 14 carbon atoms and “substituted aryl” refers to aryl groupsfurther bearing one or more substituents as set forth above.

As used herein, “heteroaryl” refers to aromatic rings containing one ormore heteroatoms (e.g., N, O, S, or the like) as part of the ringstructure, and having in the range of 3 up to 14 carbon atoms and“substituted heteroaryl” refers toheteroaryl groups further bearing oneor more substituents as set forth above.

As used herein, “alkoxy” refers to the moiety —O-alkyl-, wherein alkylis as defined above, and “substituted alkoxy” refers to alkoxyl groupsfurther bearing one or more substituents as set forth above.

As used herein, “thioalkyl” refers to the moiety —S-alkyl-, whereinalkyl is as defined above, and “substituted thioalkyl” refers tothioalkyl groups further bearing one or more substituents as set forthabove.

As used herein, “cycloalkyl” refers to ring-containing alkyl groupscontaining in the range of about 3 up to 8 carbon atoms, and“substituted cycloalkyl” refers to cycloalkyl groups further bearing oneor more substituents as set forth above.

As used herein, “heterocyclic”, refers to cyclic (i.e., ring-containing)groups containing one or more heteroatoms (e.g., N, O, S, or the like)as part of the ring structure, and having in the range of 3 up to 14carbon atoms and “substituted heterocyclic” refers to heterocyclicgroups further bearing one or more substituent's as set forth above.

“Percent Identity” means the value determined by comparing two optimallyaligned sequences over a comparison window, wherein the portion of thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) as compared to the reference sequence (whichdoes not comprise additions or deletions) for optimal alignment of thetwo sequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Optimal alignment of sequences for comparison can use any means toanalyze sequence identity (homology) known in the art, e.g., by theprogressive alignment method of termed “PILEUP” (Morrison, 1997), as anexample of the use of PILEUP); by the local homology algorithm of Smith& Waterman, (1981); by the homology alignment algorithm of Needleman &Wunsch (1970); by the search for similarity method of Pearson (1988); bycomputerized implementations of these algorithms (e.g., GAP, BEST FIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.); ClustalW (CLUSTAL inthe PC/Gene program by Intelligenetics, Mountain View, Calif., describedby, e.g., Higgins (1988); Corpet (1988); Huang (1992); and Pearson(1994); Pfamand Sonnhammer (1998); TreeAlign (Hein (1994); MEG-ALIGN,and SAM sequence alignment computer programs; or, by manual visualinspection.

Another example of an algorithm that is suitable for determiningsequence similarity is the BLAST algorithm, which is described inAltschul et al., (1990). The BLAST programs (Basic Local AlignmentSearch Tool) of Altschul, S. F., et al., (1993) searches under defaultparameters for identity to sequences contained in the BLAST “GENEMBL”database. A sequence can be analyzed for identity to all publiclyavailable DNA sequences contained in the GENEMBL database using theBLASTN algorithm under the default parameters. Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information, www.ncbi.nlm.nih.gov/; see also Zhang (1997)for the “PowerBLAST” variation. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence that either match or satisfysome positive valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., (1990)). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Extension of the word hits in each direction arehalted when: the cumulative alignment score falls off by the quantity Xfrom its maximum achieved value; the cumulative score goes to zero orbelow, due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a wordlength (W) of11, the BLOSUM62 scoring matrix (see Henikoff (1992)) alignments (B) of50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.The term BLAST refers to the BLAST algorithm which performs astatistical analysis of the similarity between two sequences; see, e.g.,Karlin (1993).

Methods of Production

As noted above, compounds or metabolites may be obtained, are obtainableor are derived from an organism having the identifying characteristicsof a B. megaterium, or alternatively from any other microorganism. Themethods comprise growing these organisms (e.g., in culture) andobtaining the compounds and/or compositions of the present invention byisolating these compounds from the culture of these organisms.

In particular, the organisms are cultivated in nutrient medium usingmethods known in the art. The organisms may be cultivated by shake flaskcultivation, small scale or large scale fermentation (including but notlimited to continuous, batch, fed-batch, or solid state fermentations)in laboratory or industrial fermentors performed in suitable medium andunder conditions allowing cell growth. The cultivation may take place insuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial sources or can be prepared according topublished compositions.

After cultivation, a supernatant, filtrate and/or extract of or derivedfrom B. megaterium may be used in formulating a pesticidal composition.

Alternatively, after cultivation, the compounds and/or metabolites maybe extracted from the culture broth.

The extract may be fractionated by chromatography. Chromatographicfractions may be assayed for toxic activity against, for example,free-living nematodes and plant parasitic nematodes, M. incognita and/orM. hapla using methods known in the art. This process may be repeatedone or more times using the same or different chromatographic methods.

Compositions

Compositions may comprise whole broth cultures, liquid cultures, orsuspensions of a strain from a B. megaterium, as well as supernatants,filtrates or extracts obtained from a strain of a B. megaterium, or thesupernatant, filtrate and/or extract or one or more metabolites orisolated compounds derived from a strain of a B. megaterium orcombinations of the foregoing which in particular have nematicidalactivity against any of the following: seed gall nematodes (Afrinawevelli), bentgrass nematodes (Anguina agrostis), shoot gall nematodes(Anguina spp.), seed gall nematodes (Anguina spp., A. amsincikiae, A.balsamophila; A. tritici), fescue leaf gall nematodes (A. graminis),ear-cockle (or wheat gall) nematodes (Anguina tritici), bud and leaf (orfoliar) nematodes (Aphelenchoides spp., A. subtenuis), begonia leaf forfern, or spring crimp, or strawberry foliar, or strawberry nematodes, orsummer dwarf) nematodes (A. fragariae), fern nematodes (A. olesistus),rice nematodes (A. oryzae), currant nematodes (A. ribes), black currant(or chrysanthemum) nematodes (A. ritzemabosi), chrysanthemum foliar orleaf nematodes (A. ritzemabosi), rice white-tip (or spring dwarf, orstrawberry bud) nematodes (A. besseyi), fungus-feeding (mushroom)nematodes (Aphelenchoides composticola), Atalodera spp. (Ataloderalonicerue, Atalodera ucri), spine nematodes (Bakernema variabile), stingnematodes (Belonolaimus spp., B. gracilis, B. longicaudatis), pine woodnematodes (Bursaphalenchus spp., B. xylophilus, B. mucronatus), sessilenematodes (Cacopaurus spp., C. epacris, C. pestis), amaranth cystnematodes (Cactodera amaranthi), birch cyst nematodes (C. betulae),cactus cyst nematodes (C. cacti), estonian cyst nematodes (C. estonica),Thorne's cyst nematodes (C. thornei), knotweed cyst nematodes (C.weissi), ring nematodes (Criconema spp.), spine nematodes (Criconemaspp., C. civellae, C. decalineatum, C. spinalineatum), ring nematodes(Criconemella axeste, C. curvata, C. macrodora, C. parva), ringnematodes (Criconemoides spp., C. citri, C. simile), spine nematodes(Crossonema fimbriatum), eucalypt cystoid nematodes (Cryphoderaeucalypti), bud, stern and bulb nematodes (Ditylenchus spp., D.angustus, D. dipsaci, D. destructor, D. intermedius), Mushroom spawnnematodes (D. myceliophagus), awl nematodes (Dolichodorus spp., D.heterocephalus, D. heterocephalous), spear nematodes (Dorylaimus spp.),stunt nematodes (Geocenamus superbus), cyst nematodes (Globodera spp.),yarrow cyst nematodes (G. achilieae), milfoil cyst nematodes (G.millefolii), apple cyst nematodes (G. mali), white cyst potato nematodes(G. pallida), golden nematodes (G. rostochiensis), tobacco cystnematodes (G. tabacum), Osborne's cyst nematodes (G. tabacumsolanacearum), horsenettie cyst nematodes (G. tabacum virginiae), pinnematodes (Gracilacus spp., G. idalimus), spiral nematodes(Helicotylenchus spp., H. africanus, H. digonicus, H. dihystera, H.erythrinae, H. multicinctus, H. paragirus, H. psendorobustus, H. solani,H. spicaudatus), sheathoid nematodes (Hermicriconemoides spp., H.biformis, H. californianus, H. chitwoodi, H. floridenisis, H. wessoni),sheath nematodes (Hemicycliophora spp., H. arenaria, H. biosphaera, H.megalodiscus, H. parvana, H. poranga, H. sheri, H. similis, H.striatula), cyst nematodes (Heterodera spp.), almond cyst nematodes (H.amygdali), oat (or cereal) cyst nematodes (H. avenue), Cajamis (orpigeon pea) cyst nematodes (H. cajani), bermudagrass (or heart-shaped,or Valentine) cyst nematodes (H. cardiolata), carrot cyst nematodes (H.carotae), cabbage cyst nematodes or brassica root eelworm (H.cruciferae), nutgrass (or sedge) cyst nematodes (H. cyperi), Japanesecyst nematodes (H. elachista), fig (or ficus, or rubber) cyst nematodes(H. fici), galeopsis cyst nematodes (H. galeopsidis), soybean cystnematodes (H. glycines), alfalfa root (or pea cyst) nematodes (Hgoettingiana), buckwheat cyst nematodes (H. graduni), barley cystnematodes (H. hordecatis), bop cyst nematodes (H. humuli), Mediterraneancereal (or wheat) cyst nematodes (H. latipons), lespedeza cyst nematodes(H. lespedezae), Kansas cyst nematodes (H. longicolla), cereals rooteelworm or oat cyst, nematodes (H. major), grass cyst nematodes (H.mani), lucerne cyst nematodes (H. inedicaginis), cyperus (or motha) cystnematodes (Heterodera mothi), rice cyst nematodes (H. oryzae), Amu-Darya(or camel thorn cyst) nematodes (H. oxiana), dock cyst nematodes (H.roSii), rumex cyst nemtodes (H. rumicis), sugar beet cyst nematodes (H.scleranthii), willow cyst nematodes (H. saliaophila), knawel cystnematodes (H. scieranthii), sowthistle cyst nematodes (H. sonchophila),tadzhik cyst nematodes (H. tadshikistanica), turkmen cyst nematodes (H.turcomanica), clover cyst nematodes (H. trifolii), nettle cyst nematodes(H. urticae), ustinov cyst nematodes (H. ustinovi), cowpea cystnematodes (H. vigni), corn cyst nematodes (H. zeae), rice root nematodes(Hirschmanniella spp., H. belli, H. candacrena, H. gracilis, H. oryzae);lance nematodes (Hopiolainuts spp.), Columbia nematodes (H. columbus),Cobb's lance nematodes (H. galeatus), crown-headed lance nematodes (H.tylenchiformis), pseudo root-knot nematodes (Hypsoperine graminis),needle nematodes (Longidorus spp., L. africanus, L. sylphus), ringnematodes (Macroposthonia (=Mesocriconema) xenoplax), cystoid nematodes(Meloidodea spp.), pine cystoid nematodes (M. florideasis), tadzhikcystoid nematodes (M. tadshikistanica), cystoid body nematodes(Meloidoderita spp.), stunt nematodes (Merlinius spp., M. brevidens, M.conicus, M. grandis, M. microdorus), root-knot nematodes (Meloidogynespp., M. acronea, M. arenaria, M brevicauda, M. camelliae, M.carolinensis, M. chitwoodi, M. exigna, M. graminicola, M. hapla, M.hispanica, M. incognita, M. incognita acrita, M. indica, M. inornata, M.javanica, M. kikuyuensis, M. konaensis, M. mail, M. microtyla, M. naasi,M. ovalis, M. platani, M, querciana, M. sasseri, M. tadshikistanica, M.thamesi), knapweed nematodes (Mesoanguina picridis), Douglas firnematodes (Nacobbodera chitwoodi), false root-knot nematodes (Nacobbusaberrans, N. batatiformis, N. dorsalis), sour paste nematodes(Panagrellus redivivus), beer nematodes (P. situsiae), needle nematodes(Paralongidorus micolaimus), spiral nematodes (Pararotyienchus spp.),stubby-root nematodes (Paratrichodorus allitus, P. minor, P. porosus, P.renifer), pin nematodes (Paratylencitus spp. P. baidaccii, P.bukowinensis, P. curvitatus, P. dianthus, P. dachistus, P. hamatus, P.holdemani, P. italiensis, P. lepidus, P. nanus, P. neoamplycephaius, P.similis), lesion (or meadow) nematodes (Pratylcnchus spp., P. alleni, P,brachyurus, P. coffeae, P. convallariac, P. crenatus, P. flakkensis, P.goodeyi, P. hexincisus, P. leiocephalus, P. minyus, P. musicola, P.neglectus, P. penetrans, P. pratensis, P. scribneri, P. thornei, P.vulnus, P. zeae), stem gall nematodes (Pterotylenchtis cecidogenus),grass cyst nematodes (Punctodera punctate), stunt nematodes(Quinisulcius acutus, Q. capitatus), burrowing nematodes (Radopholusspp.), banana-root nematodes (R. similis), rice-root nematodes (R.oryzae), red ring (or coconut, or cocopalm) nematodes (Rhadinaphelenchuscocophilus), reniform nematodes (Rotylenchtdus spp., R. reniformis, R.parvus), spiral nematodes (Rotyienchus spp., R. buxophilus, R.christiei, R. robustus), Thorne's lance nematodes (R. uniformis),Sarisodera hydrophylia, spiral nematodes (Scutellonerna spp., S.biabeum, S. brachyurum, S. bradys, S. clathricaudatum, S. christiei, S.conicephalum), grass root-gall nematodes (Subanguina radicicola), roundcystoid nematodes (Thecavermiculatus andinus), stubby-root nematodes(Trichodorus spp., T. christiei, T. kurumeensis, T. pachydermis, T.primitivus), vinegar eels (or nematodes) (Turbatrix aceti), stunt (orstylet) nematodes (Tylenchorhynchus spp., T. agri, T. annulatus, T.aspericutis, T. claytoni, T. ebriensis, T. elegans, T. golden, T.graciliformiis, T. martini, T. mashhoodi, T. microconus, T. nudus, T.oleraceae, T. penniseti, T. punensis), citrus nematodes (Tylenchulussemipenetrans), dagger nematodes (Xiphinema spp., X. americanum, X.bakeri, X. brasiliense, X. brevicolle, X. chambersi, X. coxi, X.diversicaudatum, X. index, X. insigne, X. nigerierise, X. radicicola, X.setariae, X. vulgarae, X. vuittenezi).

The compositions set forth above can be formulated in any manner.Non-limiting formulation examples include but are not limited toEmulsifiable concentrates (EC), Wettable powders (WP), Soluble liquids(SL), Aerosols, Ultra-low volume concentrate solutions (ULV), Solublepowders (SP), Microencapsulation, Water dispersed Granules, Flowables(FL), Microemulsions (ME), Nano-emulsions (NE), etc. In any formulationdescribed herein, percent of the active ingredient is within a range of0.01% to 99.99%.

The compositions may be in the form of a liquid, gel or solid. A solidcomposition can be prepared by suspending a solid carrier in a solutionof active ingredient(s) and drying the suspension under mild conditions,such as evaporation at room temperature or vacuum evaporation at 65° C.or lower.

A composition may comprise gel-encapsulated active ingredient(s). Suchgel-encapsulated materials can be prepared by mixing a gel-forming agent(e.g., gelatin, cellulose, or lignin) with a culture or suspension oflive or inactivated B. megaterium, or a cell-free filtrate or cellfraction of a B. megaterium culture or suspension, or a spray- orfreeze-dried culture, cell, or cell fraction or in a solution ofpesticidal compounds used in the method of the invention; and inducinggel formation of the agent.

The composition may additionally comprise a surfactant to be used forthe purpose of emulsification, dispersion, wetting, spreading,integration, disintegration control, stabilization of activeingredients, and improvement of fluidity or rust inhibition. In aparticular embodiment, the surfactant is a non-phytotoxic non-ionicsurfactant which preferably belongs to EPA List 4B. In anotherparticular embodiment, the nonionic surfactant is polyoxyethylene (20)monolaurate. The concentration of surfactants may range between 0.1-35%of the total formulation, preferred range is 5-25%. The choice ofdispersing and emulsifying agents, such as non-ionic, anionic,amphoteric and cationic dispersing and emulsifying agents, and theamount employed is determined by the nature of the composition and theability of the agent to facilitate the dispersion of the compositions ofthe present invention.

The composition set forth above may be combined with anothermicroorganism and/or pesticide (e.g, nematicide, fungicide,insecticide). The microorganism may include but is not limited to anagent derived from Bacillus sp. (e.g., Bacillus firmus, Bacillusthuringiensis, Bacillus pumilus, Bacillus licheniformis, Bacillusamyloliquefaciens, Bacillus subtilis), Paecilomyces sp. (P. lilacinus),Pasteuria sp. (P. penetrans), Pseudomonas sp., Brevabacillus sp.,Lecanicillium sp., Ampelomyces sp., Pseudozyma sp., Streptomyces sp (S.bikiniensis, S. costaricanus, S. avermitilis), Burkholderia sp.,Trichoderma sp., Gliocladium sp., avermectin, Myrothecium sp.,Paecilomyces spp., Sphingobacterium sp., Arthrobotrys sp.,Chlorosplrnium, Neobulgaria, Daldinia, Aspergillus, Chaetomium,Lysobacter spp, Lachnum papyraceum, Verticillium suchlasporium,Arthrobotrys oligospora, Verticillium chlamydosporium, Hirsutellarhossiliensis, Pochonia chlamydosporia, Pleurotus ostreatus, Omphalotusolearius, Lampteromyces japonicas, Brevudimonas sp., Muscodor sp.

Alternatively, the agent may be a natural oil or oil-product havingnematicidal, fungicidal and/or insecticidal activity (e.g., paraffinicoil, tea tree oil, lemongrass oil, clove oil, cinnamon oil, citrus oilincluding but not limited to bitter orange, orange, lemon; rosemary oil,pyrethrum, allspice, bergamot, blue gum, camomile, citronella, commonjasmine, common juniper, common lavender, common myrrh, field mint,freesia, gray santolina, herb hyssop, holy basil, incense tree, jasmine,lavender, marigold, mint, peppermint, pot marigold, spearmint,ylang-ylang tree, saponins). Furthermore, the pesticide may be a singlesite anti-fungal agent which may include but is not limited tobenzimidazole, a demethylation inhibitor (DMI) (e.g., imidazole,piperazine, pyrimidine, triazole), morpholine, hydroxypyrimidine,anilinopyrimidine, phosphorothiolate, quinone outside inhibitor,quinoline, dicarboximide, carboximide, phenylamide, anilinopyrimidine,phenylpyrrole, aromatic hydrocarbon, cinnamic acid, hydroxyanilide,antibiotic, polyoxin, acylamine, phthalimide, benzenoid (xylylalanine),a demethylation inhibitor selected from the group consisting ofimidazole, piperazine, pyrimidine and triazole (e.g., bitertanol,myclobutanil, penconazole, propiconazole, triadimefon, bromuconazole,cyproconazole, diniconazole, fenbuconazole, hexaconazole, tebuconazole,tetraconazole), myclobutanil, and a quinone outside inhibitor (e.g.,strobilurin). The strobilurin may include but is not limited toazoxystrobin, kresoxim-methoyl or trifloxystrobin. In yet anotherparticular embodiment, the anti-fungal agent is a quinone, e.g.,quinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether). Theanti-fungal agent may also be derived from a Reynoutria extract.

The fungicide can also be a multi-site non-inorganic, chemical fungicideselected from the group consisting of chloronitrile, quinoxaline,sulphamide, phosphonate, phosphite, dithiocarbamate, chloralkylhios,phenylpyridin-amine, cyano-acetamide oxime.

As noted above, the composition may further comprise a nematicide. Thisnematicide may include but is not limited to chemicals such asorganophosphates, carbamates, and fumigants, and microbial products suchas avermectin, Myrothecium sp. Biome (Bacillus firmus), Pasteuria spp.,Paecilomyces, and organic products such as saponins and plant oils.

The compositions may be applied using methods known in the art.Specifically, these compositions may be applied to and around plants orplant parts. Plants are to be understood as meaning in the presentcontext all plants and plant populations such as desired and undesiredwild plants or crop plants (including naturally occurring crop plants).Crop plants can be plants which can be obtained by conventional plantbreeding and optimization methods or by biotechnological and geneticengineering methods or by combinations of these methods, including thetransgenic plants and including the plant cultivars protectable or notprotectable by plant breeders' rights. Plant parts are to be understoodas meaning all parts and organs of plants above and below the ground,such as shoot, leaf, flower and root, examples which may be mentionedbeing leaves, needles, stalks, stems, flowers, fruit bodies, fruits,seeds, roots, tubers and rhizomes. The plant parts also includeharvested material, and vegetative and generative propagation material,for example cuttings, tubers, rhizomes, offshoots and seeds. Treatmentof the plants and plant parts with the compositions set forth above maybe carried out directly or by allowing the compositions to act on theirsurroundings, habitat or storage space by, for example, immersion,spraying, evaporation, fogging, scattering, painting on, injecting. Inthe case that the composition is applied to a seed, the composition maybe applied to the seed as one or more coats prior to planting the seed,or applied as a slurry or dust when planting, using one or more coatsusing methods known in the art. The seed in a particular embodiment maybe a genetically modified seed.

Plants that may be treated include but are not limited to: (A) Majoredible food crops, which include but are not limited to (1) Cereals (eAfrican rice, barley, durum wheat, einkorn wheat, emmer wheat, fingermillet, foxtail millet, hairy crabgrass, Indian barnyard millet,Japanese barnyard millet, maize, nance, oat, pearl millet, proso millet,rice, rye, sorghum, Sorghum spp., rye, spelt wheat); (2) Fruits (e.g.,abiu, acerola, achacha, African mangosteen, alpine currant, ambarella,American gooseberry, American persimmon, apple, apricot, araza, Asianpalmyra palm, Asian pear, atemoya, Australian desert raisin, avocado,azarole, babaco, bael, banana, Barbados gooseberry, bergamot, betel nut,bignay, bilberry, bilimbi, binjai, biriba, bitter orange, blackchokeberry, black mulberry, black sapote, blackberry, blue-berriedhoneysuckle, borojo, breadfruit, murmese grape, button mangosteen,cacao, calamondin, canistel, cantaloupe, cape gooseberry, cashew nut,cassabanana, cempedak, charichuelo, cherimoya, cherry, cherry of the RioGrande, cherry plum, Chinese hawthorn, Chinese white pear, chokeberry,citron, cocona, coconut, cocoplum, coffee, coffee Arabica, coffeerobusta, Costa Rica pitahaya, currants, custard apple, date, date-plum,dog rose, dragonfruit, durian, elderberry, elephant apple, Ethiopianeggplant, European nettle tree, European wild apple, feijoa, fig, gac,genipapo, giant granadilla, gooseberry, goumi, grape, grapefruit, greatmorinda, greengage, guava, hardy kiwi, hog plum, horned melon, horsemango, Indian fig, Indian jujube, jabuticaba, jackberry, jackfruit,Japanese persimmon, Japanese wineberry, jocote, jujube, kaffir lime,karanda, kei apple, kepel apple, key lime, kitembilla, kiwi fruit,korlan, kubal vine, kuwini mango, kwai muk, langsat, large cranberry,lemon, Liberian coffee, longan, loquat, lychee, malay apple, mameysapote, mammee apple, mango, mangosteen, maprang, marang, medlar, melon,Mirabelle plum, miracle fruit, monkey jack, moriche palm, mountainpapaya, mountain soursop, mulberry, naranjilla, natal plum, northernhighbush blueberry, olive, otaheite gooseberry, oval kumquat, papaya,para guava, passionfruit, pawpaw, peach, peach-palm, pear, pepino,pineapple, pitomba Eugenia luschnathiana, pitomba talisia esculenta,plantain, plum, pomegranate, pomelo, pulasan, purple chokeberry, quince,rambutan, ramontchi, raspberry, red chokeberry, red currant, redmulberry, red-fruited strawberry guava, rhubarb, rose apple, roselle,safou, salak, salmonberry, santol, sapodilla, satsuma, seagrape,soncoya, sour cherry, soursop, Spanish lime, Spanish tamarind, starapple, starfruit, strawberry, strawberry guava, strawberry tree, sugarapple, Surinam cherry, sweet briar, sweet granadilla, sweet lime,tamarillo, tamarind, tangerine, tomatillo, tucuma palm, Vaccinium spp.,velvet apple, wampee, watermelon, watery rose apple, wax apple, whitecurrant, white mulberry, white sapote, white star apple, wolfberry(Lyceum barbarum, L. chinense), yellow mombin, yellow pitaya,yellow-fruited strawberry, guava, (3) Vegetables (e.g., ackee, agate,air potato, Amaranthus spp., American groundnut, antroewa, armeniancucumber, arracacha, arrowleaf elephant ear, arrowroot, artichoke, ashgourd, asparagus, avocado, azuki bean, bambara groundnut, bamboo,banana, barbados gooseberry, beet, beet root, bitter gourd, bittervetch, bitterleaf, black mustard, black radish, black salsify, blanchedcelery, breadfruit, broad bean, broccoli, brussels sprout, Buck's hornplantain, buttercup squash, butternut squash, cabbage, caigua, calabash,caraway seeds, carob, carrot, cassabanana, cassava, catjang,cauliflower, celeriac, celery, celtuce, chard, chayote, chickpea,chicory, chilacayote, chili pepper (Capsicum annuum, C. baccatum, C.chinense, C. frutescens, C. pubescens), Chinese cabbage, Chinese waterchestnut, Chinese yam, chives, chufa sedge, cole crops, common bean,common purslane, corn salad, cowpea, cress, cucumber, cushaw pumpkin,drumstick tree, eddoe, eggplant, elephant foot yam, elephant garlic,endive, enset, Ethiopian eggplant, Florence fennel, fluted gourd, gac,garden rocket, garlic, geocarpa groundnut, good king henry, grass pea,groundut, guar bean, horse gram, horseradish, hyacinth bean, iceplant,Indian fig, Indian spinach, ivy gourd, Jerusalem artichoke, jicama,jute, kale, kohlrabi, konjac, kurrat, leek, lentil, lettuce, Lima bean,lotus, luffa, maca, maize, mangel-wurzel, mashua, moso bamboo, mothbean, mung bean, napa cabbage, neem, oca, okra, oldham's bamboo, olive,onion, parsnip, pea, pigeon pea, plantain, pointed gourd, potato,pumpkins, squashes, quinoa, radish, rapeseed, red amaranth, rhubarb,ribbed gourd, rice bean, root parsley, runner bean, rutabaga, sago palm,salsify, scallion, sea kale, shallot, snake gourd, snow pea, sorrel,soybean, spilanthes, spinach, spinach beet, sweet potato, taro, tarwi,teasle gourd, tepary bean, tinda, tomato, tuberous pea, turnip,turnip-rooted chervil, urad bean, water caltrop trapa bicornis, watercaltrop trapa natans, water morning slory, watercress, welsh onion, westAfrican okra, west Indian gherkin, white goosefoot, white yam, wingedbean, winter purslane, yacón, yam, yard-long bean, zucchinietables); (4)Food crops (e.g., abiu, acerola, achacha, ackee, African mangosteen,African rice, agate, air potato, alpine currant, Amaranthus app.,Ambarrella, American gooseberry, American groundnut, American persimmon,antroewa, apple, apricot, arazá, Armenian cucumber, arracacha, arrowleafelephant ear, arrowroot, artichoke, ash gourd, Asian palmyra palm, Asianpear, asparagus, atemoya, Australian desert raisin, avocado, azarole,azuki bean, babaco, bael, bambara groundnut, bamboo, banana, barbadosgooseberry, barley, beet, beetroot, bergamot, betel nut, bignay,bilberry, bilimbi, binjai, biriba, bitter gourd, bitter orange, bittervetch, bitterleaf, black chokeberry, black currant, black mulberry,black mustard, black radish, black salsify, black sapote, blackberry,blanched celery, blue-berried honeysuckle, borojo, breadfruit, broadbean, broccoli, Brussels sprout, Buck's horn plantain, buckwheat,Burmese grape, buttercup squash, butternut squash, button mangosteen,cabbage, cacao, caigua, calabash, calamondin, canistel, cantaloupe, capegooseberry, caraway seeds, carob, carrot, cashew nut, cassaya, catjang,cauliflower, celeriac, celery, celtuce, cempedak, chard, charichuelo,chayote, cherimoya, cherry, cherry of the Rio Grande, cherry plum,chickpea, chicory, chilacayote, chili pepper (Capsicum annuum, C.baccatum, C. chinense, C. frutescens, C. pubescens), Chinese cabbage,Chinese hawthorn, Chinese water chestnut, Chinese white pear, Chineseyam, chives, chokeberry, chufa sedge, citron, cocona, coconut, cocoplum,coffee, coffee (Arabica and Robusta types), cole crops, common bean,common purslane, corn salad, Costa Rica pitahaya, cowpea, cress,cucumber, currants, cushaw pumpkin, custard apple, date, date-plum, dogrose, dragonfruit, drumstick tree, durian, durum wheat, eddoe, eggplant,einkorn wheat, elderberry, elephant apple, elephant foot yam, elephantgarlic, emmer wheat, endive, enset, Ethiopian eggplant, European nettletree, European wild apple, feijoa, fig, finger millet, florence fennel,fluted gourd, foxtail millet, gac, garden rocket, garlic, genipapo,geocarpa groundut, giant granadilla, good king henry, gooseberry, goumi,grape, grapefruit, grass pea, great morinda, greengage, groundnut,grumichama, guar bean, guava, hairy crabgrass, hardy kiwi, hog plum,horned melon, horse gram, horse mango, horseradish, hyacinth bean,iceplant, Indian barnyard millet, Indian fig, Indian jujube, Indianspinach, ivy gourd, jabuticaba, jackalberry, jackfruit, jambul, Japanesebarnyard millet, Japanese persimmon, Japanese wineberry, Jerusalemartichoke, jocote, jujube, jute, kaffir lime, kale, karanda, kei apple,kepel apple, key lime, kitembilla, kiwifruit, kohlrabi, konjac, korlan,kubal vine, kurrat, kuwini mango, kwai muk, langsat, large cranberry,leek, lemon, lentil, lettuce, Liberian coffee, lima bean, longan,loquat, lotus, luffa, lychee, maca, maize, malay apple, mamey saptoe,mammee apple, mangel-wurzel, mango, mangosteen, maprang, marang, mashua,medlar, melon, Mirabelle plum, miracle fruit, monk fruit, monkey jack,moriche palm, moso bamboo, moth bean, mountain papaya, mountain soursop,mulberry, mung bean, mushrooms, nance, napa cabbage, naranjilla, natalplum, neem, northern highbush blueberry, oat, oca, oil palm, okra,oldman's bamboo, olive, onion, orange, otaheite gooseberry, ovalkumquat, papaya, para guava, parsnip, passionfruit, pawpaw, pea, peach,peach-palm, pear, pearl millet, pepino, pigeon pea, pineapple, Pitomba(Eugenia luschnathiana, Talisia esculenta), plantain, plum, pointedgourd, pomegranate, pomelo, potato, proso millet, pulasan, pumpkins andsquashes, purple chokeberry, quince, quinoa, radish, rambutan,ramontchi, rapeseed, raspberry, red amaranth, red chokeberry, redcurrant, red mulberry, red-fruited strawberry guava, rhubarb, ribbedgourd, rice, rice bean, root parsley, rose apple, roselle, runner bean,rutabaga, rye, safou, sago palm, salak, salmonberry, salsify, santol,sapodilla, Satsuma, scallion, sea kale, seagrape, shallot, snake gourd,snow pea, soncoya, sorghum, Sorghum spp., sorrel, sour cherry, soursop,soybean, Spanish lime, Spanish tamarind, spelt wheat, spilanthes,spinach, spinach beet, star apple, starfruit, strawberry, strawberryguava, strawberry tree, sugar apple, sugar beet, sugarcane, surinamcherry, sweet briar, sweet granadilla, sweet lime, sweet potato,tamarillo, tamarind, tangerine, taro, tarwi, teasle gourd, tef, teparybean, tinda, tomatillo, tomato, tuberous pea, tucuma palm, turnip,turnip-rooted chervil, urad bean, Vaccinium spp., velvet apple, wampee,water caltrop (Trapa bicornis, T. natans), water morning glory,watercress, watermelon, watery rose apple, wax apple, welsh onion, westAfrican okra, west Indian gherkin, wheat, white currant, whitegoosefoot, white mulberry, white sapote, white star apple, white yam,winged bean, winter purslane, wolfberry (Lycium barbarum, L. chinense),yacón, yam, yangmei, yard-long bean, yellow mombin, yellow pitaya,yellow-fruited strawberry guava, zucchini; (B) Other edible crops, whichincludes but is not limited to (1) Herbs (e.g., Absinthium, alexanders,basil, bay laurel, betel nut, camomile, chervil, chili pepper (Capsicumannuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), chilipeppers, chives, cicely, common rue, common thyme, coriander, cress,culantro, curly leaf parsley, dill, epazote, fennel, flat leaf parsley,ginseng, gray santolina, herb hyssop, holy basil, hop, jasmine, kaffirlime, lavender, lemon balm, lemon basil, lemon grass, lovage, marjoram,mint, oregano, parsley, peppermint, perilla, pot marigold, rooibos,rosemary, sage, shiny-leaft buckthorn, sorrel, spearmint, summer savory,tarragon, That basil, valerian, watercress, wild betel, winter savory,yerba mate); (2) Spices (e.g., ajowan, allspice, anise, bay laurel,black cardamom, black mustard, black pepper, caper, caraway seeds,cardamom, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C.frutescens, C. pubescens), chili peppers, cinnamon, clove, commonjuniper, coriander, cumin, fennel, fenugreek, garlic, ginger, kaffirlime, liquorice, nutmeg, oregano, pandan, parsley, saffron, star anise,turmeric, vanilla, white mustard); (2) Medicinal plants (e.g.,absinthium, alfalfa, aloe vera, anise, artichoke, basil, bay laurel,betel leat, betel nut, bilberry, black cardamom, black mustard, blackpepper, blue gum, borojo, camomlie, caper, cardamom, castor bean, chilipeppers, Chinese yam, chives, cola nut, common jasmine, common lavender,common myrrh, common rue, cilantro, cumin, dill, dog rose, epazote,fennel, fenugreek, gac, garlic, ginger, gray santolina, gum Arabic, herbhyssop, holy basil, horseradish, incense tree, lavender, lemon grass,liquorice, lovage, marijuana, marjoram, monk fruit, neem, opium,oregano, peppermint, pot marigold, quinine, red acacia, red currant,rooibos, safflower, sage, shiny-leaf buckthorn, sorrel, spilanthes, staranise, tarragon, tea, turmeric, valerian, velvet bean, watercress, whitemustard, white sapote, wild betel, wolfberry (Lycium barbarum, L.chinense), yerba mate); (3) Stimulants (e.g., betel leaf, betel nut,cacao, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C.frutescens, C. pubescens), chili peppers, coffee, coffee (Arabica,Robusta), cola nut, khat, Liberian coffee, tea, tobacco, wild betel,yerba mate); (4) Nuts (e.g., almond, betel nut, Brazil nut, cashew nut,chestnut, Chinese water chestnut, coconut, cola nut, common walnut,groundnut, hazelnut, Japanese stone oak, macadamia, nutmeg, paradisenut, pecan nut, pistachio nut, walnut); (5) Edible seeds (e.g., blackpepper, Brazil nut, chilacayote, cola nut, fluted gourd, lotus, opium,quinoa, sesame, sunflower, water caltrop (Trapa bicornis, T. natans);(6) Vegetable oils (e.g., black mustard, camelina, castor bean, coconut,cotton, linseed, maize, neem, niger seed, oil palm, olive, opium,rapeseed, safflower, sesame, soybean, sunflower, tung tree, turnip); (7)Sugar crops (e.g., Asian palmyra palm, silver date palm, sorghum, sugarbeet, sugarcane); (8) Pseudocereals (e.g., Amaranthus spp., buckwheat,quinoa, red amaranth); (9) Aphrodisiacs (e.g., borojo, celery, durian,garden rocket, ginseng, maca, red acacia, velvet bean); (C) Non foodcategories, including but not limited to (1) forage and dodder crops(e.g., agate, alfalfa, beet, broad bean, camelina, catjang, grass pea,guar bean, horse gram, Indian barnyard millet, Japanese barnyard millet,lespedeza, lupine, maize, mangel-wurzel, mulberry, niger seed, rapeseed,rice bean, rye); (2) Fiber crops (e.g., coconut, cotton, fique, hemp,henequen, jute, kapok, kenaf, linseed, manila hemp, New Zealand flax,ramie, roselle, sisal, white mulberry); (3) Energy crops (e.g., bluegum, camelina, cassaya, maize, rapeseed, sorghum, soybean, Sudan grass,sugar beet, sugarcane, wheat); (4) Alcohol production, (e.g., barley,plum, potato, sugarcane, wheat, sorghum); (5) Dye crops (e.g., chayroot, henna, indigo, old fustic, safflower, saffron, turmeric); (6)Essential oils (e.g., allspice, bergamot, bitter orange, blue gum,camomile, citronella, clove, common jasmine, common juniper, commonlavender, common myrrh, field mint, freesia, gray santolina, herbhyssop, holy basil, incense tree, jasmine, lavender, lemon, marigold,mint, orange, peppermint, pot marigold, spearmint, ylang-ylang tree);(6) Green manures (e.g., alfalfa, clover, lacy Phacelia, sunn hemp,trefoil, velvet bean, vetch); (7) Erosion prevention (e.g., bamboo,cocoplum; (8) Soil improvement (e.g., lupine, vetch); (9) Cover crops(e.g., Alfalfa, lacy Phacelia, radish); (10) Botanical pesticides (e.g.,jicama, marigold, neem, pyrethrum); (11) Cut flowers (e.g., carnation,chrysanthemum, daffodil, dahlia, freesia, gerbera, marigold, rose,sunflower, tulip); (12) Ornamental plants (e.g., African mangosteen,aloe vera, alpine currant, aster, black chokeberry, breadfruit,calamondin, carnation, cassabanana, castor bean, cherry plum,chokeberry, chrysanthemum, cocoplum, common lavender, crocus, daffodil,dahlia, freesia, gerbera, hyacinth, Japanese stone oak, Jasmine, lacyPhacelia, lotus, lupine, marigold, New Zealand flax, opium, purplechokeberry, ramie, red chokeberry, rose, sunflower, tulip, whitemulberry); (D) Trees which include but are not limited to abelia.almond. apple, apricot. arborvitae nigra american, arborvitae, ash,aspen, azalea, baidcypress, beautybush, beech, birch, black tupelo,blackberry, blueberry, boxwood, buckeye, butterfly bush, butternut,camellia, catalpa, cedar, cherry, chestnut, coffeetree, crab trees,crabapple, crapemyrtle, cypress, dogwood, douglasfir, ebony, elderAmerican, elm, fir, forsythia, ginkgo, goldenraintree, hackberry,hawthorn, hazelnut, hemlock, hickory, holly, honeylocust, horsechestnut,hydrangea, juniper, lilac, linden, magnolia, maple, mockorange,mountainash, oak, olive, peach, pear, pecan, pine, pistache, planetree,plum, poplar, pivet, raspberry, redbud, redcedar, redwood, rhododendron,rose-of-sharon, sassafras, sequoia, serviceberry, smoketree, soapberry,sourwood, spruce, strawberry tree, sweetshrub, sycamore, tuliptree,viburnum, walnut, weigela, willow, winterberry, witchhazel, zelkova; (E)Turf which includes but is not limited to Kentucky bluegrass, tallfescue, Bermuda grass, zoysia grass, perennial ryegrass, fine fescues(e.g.; creeping red, chewings, hard, or sheep fescue).

The compositions may also be applied to the soil using methods known inthe art (see, for example, Chitwood, “Nematicides”, available atnaldc.nal.usda.gov/download/43874/PDF. Such methods include but are notlimited to fumigation, drip irrigation or chemigation, soilincorporation, soil drenching, seed treatment and dressing, bare rootdip.

Pesticidal Uses

The compositions, cultures, supernatants, metabolites and pesticidalcompounds set forth above may be used as pesticides. In particular, thecompositions, cultures, supernatants, metabolites and pesticidalcompounds as set forth above may be used as insecticides andnematicides, alone or in combination with one or more pesticidalsubstances set forth above. Specifically, nematodes that may becontrolled using the method set forth above include but are not limitedto free living nematodes, parasitic nematodes such as root-knot, cyst,and lesion nematodes, including but not limited to seed gall nematodes(Afrina wevelli) bentgrass nematodes (Anguina agrostis), shoot gallnematodes (Anguina spp.), seed gall nematodes (Anguina spp., A.amsinckiae, A. balsamophila; A. tritici), fescue leaf gall nematodes (A.graminis), ear-cockle (or wheat gall) nematodes (Anguina tritici), budand leaf (or foliar) nematodes (Aphelenchoides spp., A. subtenuis),begonia leaf (or fern, or spring crimp, or strawberry foliar, orstrawberry nematodes, or summer dwarf) nematodes (A. fragariae), fernnematodes (A. olesistus), rice nematodes (A. oryzae), currant nematodes(A. ribes), black currant (or chrysanthemum) nematodes (A. ritzemabosi),chrysanthemum foliar or leaf nematodes (A. ritzemabosi), rice white-tip(or spring dwarf, or strawberry bud) nematodes (A. besseyi),fungus-feeding (mushroom) nematodes (Aphelenchoides composticola),Atalodera spp. (Atalodera lonicerae, Atalodera ucri), spine nematodes(Bakernema variabile), sting nematodes (Belonolainuis spp., B. gracilis,B. langicaudatus), pine wood nematodes (Bursaphalenchus spp., B.xylophilus, B. mucronauts), sessile nematodes (Cacopaurus spp., C.epacris, C. pestis), amaranth cyst nematodes (Cactodera amaranthi),birch cyst nematodes (C. betulae), cactus cyst nematodes (C. cacti),estonian cyst nematodes (C. estonica), Thorne's cyst nematodes (C.thornei), knotweed cyst nematodes (C. weissi), ring nematodes (Criconemaspp.), spine nematodes (Cricanerna spp., C. civellae, C. decalineatum,C. spinalineatum), ring nematodes (Criconemella axeste, C. curvata, C.macrodora, C. parva), ring nematodes (Criconemoides spp., C. citri, C.simile), spine nematodes (Crossonema fimbriatum), eucalypt cystoidnematodes (Cryphodera eucalypti), bud, stem and bulb nematodes(Ditylenchus spp., D. angustus, D. dipsaci, D. destructor, D.intermedius), Mushroom spawn nematodes (D. myceliophagus), awl nematodes(Dolichodonts spp., D. heterocephalus, D. heterocephalous), spearnematodes (Dorylaimus spp.), stunt nematodes (Geocenamus superbus), cystnematodes (Globodera spp.), yarrow cyst nematodes (G. achilleae),milfoil cyst nematodes (G. millefolii), apple cyst nematodes (G. mali),white cyst potato nematodes (G. pallida), golden nematodes (G.rostochiensis), tobacco cyst nematodes (G. tabacum), Osborne's cystnematodes (G. tabacum solanacearum), horsenettle cyst nematodes (G.tabacum virginiae), pin nematodes (Gracilactts spp., G. idalimus),spiral nematodes (Helicotylenchus spp., H. africanus, H. digonicus, H.dihystera, H. erythrinae, H. muiticinctus, H. paragirus, H.pseudorobustus, H. solani, H. spicaudatus), sheathoid nematodes(Hemicriconemoides spp., H. biformis, H. cakformianus, H. chitwoodi, H.florideasis, H. wessoni), sheath nematodes (Henticycilophora spp. H.arenaria, H. biosphaera, H. megalodiscus, H. parvana, H. poranga, H.sheri, H. similis, H. striatula), cyst nematodes (Heterodera spp.),almond cyst nematodes (H. amygdali), oat (or cereal) cyst nematodes (H.avenae), Cajanus (or pigeon pea) cyst nematodes (H. cajani),bermudagrass or heart-shaped, or Valentine) cyst nematodes (H.cardiolata), carrot cyst nematodes (H. carotae), cabbage cyst nematodesor brassica root eelworm (H. cruciferae), nutgrass or sedge) cystnematodes (H. cyperi), Japanese cyst nematodes (H. elachista), fig (orficus, or rubber) cyst nematodes (H. fici), galeopsis cyst nematodes (H.galeopsidis), soybean cyst nematodes (H. glycines), alfalfa root (or peacyst) nematodes (H. goettingiana), buckwheat cyst nematodes (H.graduni), barley cyst nematodes (H. hordecalis), hop cyst nematodes (H.hamuli), Mediterranean cereal (or wheat) cyst nematodes (H. latipons),lespedeza cyst nematodes (H. lespedezae), Kansas cyst nematodes (H.longicolla), cereals root eelworm or oat cyst nematodes (H. major),grass cyst nematodes (H. mani), lucerne cyst nematodes (H. medicaginis),cyperus (or motha) cyst nematodes (Heterodera mothi), rice cystnematodes (H. oryzae), Amu-Darya (or camel thorn cyst) nematodes (H.oxiana), dock cyst nematodes (H. rosii), rumex cyst nemtodes (H.rumicis), sugar beet cyst nematodes (H. schachtii), willow cystnematodes (H. salixophila), knawel cyst nematodes (H. scleranthii),sowthistle cyst nematodes (H. sonchophila), tadzhik cyst nematodes (H.tadshikistanica), turkmen cyst nematodes (H. turcomanica), clover cystnematodes (H. trifolii), nettle cyst nematodes (H. urticae), ustinovcyst nematodes (H. ustinovi), cowpea cyst nematodes (H. vigni), corncyst nematodes (H. zeae), rice root nematodes (Hirschmanniella spp., H.belli, H. caudacena, H. gracilis, H. oryzae), lance nematodes(Hoplolaimus spp.), Columbia nematodes (H. columbus), Cobb's lancenematodes (H. galeatus), crown-headed lance nematodes (H,tylenchiformis), pseudo root-knot nematodes (Hypsoperine graminis),needle nematodes (Longidorus spp., L. africanus, L sylphus), ringnematodes (Macroposthonia (=Mesocriconema) xenoptax), cystoid nematodes(Meloidodera spp.), pine cystoid nematodes (M. floridensis), tadzhikcystoid nematodes (M. tadshikistanica), cystoid body nematodes(Meloidoderita spp.), stunt nematodes (Merlinius spp., M. brevidens, M.conicus, M. grandis, M. microdorus), root-knot nematodes (Meloidogynespp., M. acronea, M. arenaia, M. artiellia, M. brevicauda, M. camelliae,M. carolinensis, M. chitwoodi, M. exigua, M. graminicola, M. hapla, M.hispanica, M. incognita, M. incognita acrita, M. indica, M. inornata, M.javanica, M. kikuyuensis, M. konaensis, M. mali, M. microtyla, M. naasi,M. ovalis, M. platani, M. querciana, M. sasseri, M. tadshikistanica, M.thamesi), knapweed nematodes (Mesoanguina picridis), Douglas firnematodes (Nacobbodera chitwoodi), false root-knot nematodes (Nacobbusaberrans, N. batatiformis, N. dorsalis), sour paste nematodes(Panagrellus redivivus), beer nematodes (P. silusiae), needle nematodes(Paralongidorus microlaimus), spiral nematodes (Pararotyienchus spp.),stubby-root nematodes (Paratrichodorus allius, P. minor, P. porous, P.renifer), pin nematodes (Paratylenchus spp., P. baldaccii, P.bukowinensis, P. curvitatus, P. dianthus, P. elachistus, P. hamatus, P.holdemani, P. italiensis, P. lepidus, P. nanus, P. neoamplycephalus, P.similis), lesion (or meadow) nematodes (Pratylenchus spp., P. alleni, P.brachyurus, P. coffeae, P. convallariae, P. crenatus, P. flakkensis, P.goodeyi, P. hexincisus, P. leiocephalus, P. minyus, P. musicola, P.neglectus, P. penetrans, P. pratensis, P. scribneri, P. thornei, P.vulnus, P. zeae), stem gall nematodes (Pterotylenchus cecidogenus),grass cyst nematodes (Punctodera punctate), stunt nematodes(Quinisulcius acutus Q. capitatus), burrowing nematodes (Radopholusspp.), banana-root nematodes (R. similis), rice-root, nematodes (R.oryzae), red ring (or coconut, or cocopalm) nematodes (Rhadinaphelenchuscocophilus), reniform nematodes (Rotylenchulus spp., R. reniformis, R.parvus), spiral nematodes (Rotylenchus spp., R. buxophilus, R.christiei, R. robustus), Thorne's lance nematodes (R. uniformis),Sarisodera hydrophylla, nematodes (Sctuellonema spp., S. blaberum, S.brachyurum, S. bradys, S. clathricaudatum, S. christiei, S.conicephalum), grass root-gall nematodes (Subanguina radicicola), roundcystoid nematodes (Thecavermiculatus andinus), stubby-root nematodes(Trichodorus spp., T. christiei, T. kurumeensis, T. pachydermis, T.primitivus), vinegar eels (or nematodes) (Turbatrix aceti), stunt (orstylet) nematodes (Tylenchorhynchus spp., T. agri, T. annulatus, T.aspericutis, T. claytoni, T. ebriensis, T. elegans, T. golden, T.graciliformis, T. martini, T. mashhoodi, T. microconus, T. nudus, T.oleraceae, T. penniseti, T. punensis), citrus nematodes (Tylenchulussemipenetrans), dagger nematodes (Xiphinema spp., X. americanum, X.bakeri, X. brasiliense, X. brevicolle, X. chambersi, X. coxi, X.diversicaudatum, X. index, X. insigne, X. nigeriense, X. radicicola, X.setariae, X. vulgarae, X, vuittenezi).

Application of an effective pesticidal control amount of a supernatant,filtrate or extract containing a pesticidally active metabolite, orisolated compound produced by the B. megaterium or application ofcombinations of the foregoing is provided. The strain or supernatant orfiltrate or extract, metabolite and/or compound is applied, alone or incombination with another pesticidal substance, in an effective pestcontrol or pesticidal amount. An effective amount is defined as thatquantity of microorganism cells, supernatant, filtrate or extract,metabolite and/or compound alone or in combination with anotherpesticidal substance that is sufficient to modulate pest infestation.The effective rate can be affected by pest species present, stage ofpest growth, pest population density, and environmental factors such astemperature, wind velocity, rain, time of day and seasonality. Theamount that will be within an effective range in a particular instancecan be determined by laboratory or field tests.

Plant Growth Promotion

The compositions disclosed herein, in particular, Bacillus megateriumand/or a supernatant, filtrate, extract, compound, metabolite or cellfraction obtained from a culture of B. megaterium, can be used tomodulate or more particularly promote growth of plants, e.g. crops suchas fruit (e.g., strawberry), vegetable (e.g., tomato, squash, pepper,eggplant), or grain crops (e.g., soy, wheat, rice, corn), tree, flower,ornamental plants, shrubs (e.g., cotton, roses), bulb plant (e.g.,onion, garlic) or vine (e.g., grape vine). The compositions can also beused to modulate the germination of a seed(s) in a plant(s).

The compositions disclosed herein, or formulated product, can be usedalone or in combination with one or more other components as describedbelow, such as growth promoting agents and/or anti-phytopathogenicagents in a tank mix or in a program (sequential application calledrotation) with predetermined order and application interval during thegrowing season. When used in a combination with the above-mentionedproducts, at a concentration lower than recommended on the productlabel, the combined efficacy of the two or more products (one of whichis the said composition disclosed herein) is, in certain embodiments,greater than the sum of each individual component's effect. Hence, theeffect is enhanced by synergism between these two (or more) products,and the risk for the development of pesticide resistance among the plantpathogenic strains is reduced.

The composition can be applied by root dip at transplanting,specifically by treating a fruit or vegetable with the composition bydipping roots of the fruit or vegetable in a suspension of saidcomposition (about 0.25 to about 1.5% and more particularly about 0.5%to about 1.0% by volume) prior to transplanting the fruit or vegetableinto the soil.

Alternatively, the composition can be applied by drip or otherirrigation system. Specifically, the composition can be injected into adrip irrigation system. In a particular embodiment, the composition isapplied at a concentration of 1×10⁸ CFU/ml in a volume of approximately11 to approximately 4 quarts per acre.

In yet another embodiment, the composition can be added as an in-furrowapplication. Specifically, the composition can be added as an in-furrowspray at planting using nozzles calibrated to deliver a total output of2-6 gallons/acre. Nozzles are placed in the furrow opener on the planterso that the pesticide application and seed drop into the furrow aresimultaneous.

Mixtures of the disclosed compositions with, for example, a solid orliquid adjuvant are prepared in known manner. For example, mixtures canbe prepared by homogeneously mixing and/or grinding the activeingredients with extenders such as solvents, solid carriers and, whereappropriate, surface-active compounds (surfactants). The compositionscan also contain additional ingredients such as stabilizers, viscosityregulators, binders, adjuvants as well as fertilizers or other activeingredients in order to obtain special effects.

Combinations with Plant Growth Promoting Agents

The compositions disclosed herein can be used in combination with othergrowth promoting agents such as synthetic or organic fertilizers (e.g.,di-ammonium phosphate, in either granular or liquid form), compost teas,seaweed extracts, plant growth hormones such as IAA (indole acetic acid)used in a rooting hormone treatment for transplants either alone or incombination with plant growth regulators such as IBA (indole butyricacid) and NAA (naphthalene acetic acid), and growth promoting microbes,such as, for example, Bacillus spp., Pseudomonads, Rhizobia, andTrichoderma.

Seed Treatments

Seed treatments include application of a pesticidal composition,optionally in combination with other bioactive, antagonistic orsymbiotic agents to the surface of a seed prior to sowing. Pesticidaltoxins, proteins, and/or compounds disclosed herein can be applied toseeds as dry powders, slurried powders or sprayed on the seed beforeplanting.

The compositions disclosed herein can be formulated for seed treatmentsin any of the following modes: dry powder, water slurriable powder,liquid solution, flowable concentrate or emulsion, emulsion,microcapsules, gel, or water dispersible granules.

In the case of a dry powder, the active ingredient is formulatedsimilarly to a wettable powder, but with the addition of a stickingagent, such as mineral oil, instead of a wetting agent. For example, onekg of purified talc powder (sterilized for 12 h), 15 g calciumcarbonate, and 10 g carboxymethyl cellulose are mixed under asepticconditions following the method described by Nandakumar et al (2001).Active ingredient(s) is/are mixed in a 1:2.5 ratio (suspension to drymix) and the product is shade dried to reduce moisture content to20-35%.

The compositions disclosed herein can also be used in combination withseed-coating agents. Such seed coating agents include, but are notlimited to, ethylene glycol, polyethylene glycol, chitosan,carboxymethyl chitosan, peat moss, resins and waxes or chemicalfungicides or bactericides with either single site, multisite or unknownmode of action.

In additional embodiments, the disclosed compositions can be applied toseeds by seed imbibition or as a powdered inoculum.

Anti-Phytopathogenic Agents

The compositions disclosed herein can also be used in combination withother anti-phytopathogenic agents, such as plant extracts,biopesticides, inorganic crop protectants (such as copper), surfactants(such as rhamnolipids; Gandhi et al., 2007) or natural oils such asparaffinic oil and tea tree oil possessing pesticidal properties orchemical fungicides or bactericides with either single site, multisiteor unknown mode of action. As defined herein, an “anti-phytopathogenicagent” is an agent that modulates the growth of a plant pathogen,particularly a pathogen causing soil-borne disease on a plant, oralternatively prevents infection of a plant by a plant pathogen. A plantpathogen includes but is not limited to a fungus, bacteria, actinomyceteor virus.

As noted above, the anti-phytopathogenic agent can be a single-siteanti-fungal agent which can include but is not limited to benzimidazole,a demethylation inhibitor (DMI) (e.g., imidazole, piperazine,pyrimidine, triazole), morpholine, hydroxypyrimidine, anilinopyrimidine,phosphorothiolate, quinone outside inhibitor, quinoline, dicarboximide,carboximide, phenylamide, anilinopyrimidine, phenylpyrrole, aromatichydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin,acylamine, phthalimide, benzenoid (xylylalanine). In a more particularembodiment, the antifungal agent is a demethylation inhibitor selectedfrom the group consisting of imidazole, piperazine, pyrimidine andtriazole (e.g., bitertanol, myclobutanil, penconazole, propiconazole,triadimefon, bromuconazole, cyproconazole, diniconazole, fenbuconazole,hexaconazole, tebuconazole, tetraconazole). In a most particularembodiment, the antifungal agent is myclobutanil. In yet anotherparticular embodiment, the antifungal agent is a quinone outsideinhibitor (e.g., strobilurin). The strobilurin may include but is notlimited to azoxystrobin, kresoxim-methyl or trifloxystrobin. In yetanother particular embodiment, the anti-fungal agent is a quinone, e.g.,quinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether).

In yet a further embodiment, the fungicide is a multi-sitenon-inorganic, chemical fungicide selected from the group consisting ofchloronitrile, quinoxaline, sulphamide, phosphonate, phosphite,dithiocarbamate, chloralkylhios, phenylpyridine-amine, andcyano-acetamide oxime.

In yet a further embodiment, the anti-phytopathogenic agent can bestreptomycin, tetracycline, oxytetracycline, copper, or kasugamycin.

EXAMPLES

The composition and methods set forth above will be further illustratedin the following, non-limiting Examples. The examples are illustrativeof various embodiments only and do not limit the claimed inventionregarding the materials, conditions, weight ratios, process parametersand the like recited herein.

Example 1 Isolation and Identification of the Microbes

Bacillus megaterium strain H491 was isolated from soil collected inKwadaso, Ghana, Africa. Bacillus megaterium strains M018 and J142 wererecovered from soils collected in California. The bacteria wererecovered from the sample by traditional plate dilution methods asdescribed by Lorch et al., 1995. Briefly, the sample was resuspended insterile deionized water. Serial dilutions of the resuspended sample wereprepared in sterile water. Some of these dilutions were spread onto agarplates (for example Potato Dextrose Agar) and incubated in the dark andat room temperature. After several days of incubation, colonies wererecovered from the surface of the agar plate.

The isolates grew as dense, flat, cream colored colonies. The isolatedbacteria are gram-positive.

Example 2 Identification of Bacillus megaterium Strain H491, StrainJ142, and Strain M018 by Sequencing of rRNA and recA Genes

The isolates (H491, J142 and M018) were identified as a Bacillusmegaterium through 16s rRNA amplification and additional sequencing ofthe recA gene using universal bacterial primers (Cerritos et al., 2008).

Growth from a 24 hour potato dextrose plate was scraped with a sterileloop and resuspended in DNA extraction buffer. DNA was extracted usingthe MoBio Ultra Clean Microbial DNA extraction kit. DNA extract waschecked for quality/quantity by electrophoresis of a 5 uL aliquot on a1% agarose gel.

rRNA Sequences

PCR reactions for the amplification of the 16S rRNA gene were set up bycombining 2 mL of the clean DNA extract with 25 mL of GoTaq GreenMastermix, 1.5 mL forward primer (27F primer; 5′-AGAGTTTGATCMTGGCTCAG-3′(SEQ ID NO:10)), 1.5 mL reverse primer (1525R primer;5′-AAGGAGGTGWTCCARCC-3′ (SEQ ID NO:11)). The reaction volume was made upto 50 mL using sterile nuclease-free water. The PCR reaction wasconducted on a thermocycler machine under the following conditions: 10minutes at 95° C. (initial denaturing), 30 cycles of 45 seconds at 94°C., 45 seconds at 55° C. and 2 minutes at 72° C., followed by 5 minutesat 72° C. (final extension) and a final hold temperature of 10° C.

The size, quality and quantity of the PCR product was evaluated byelectrophoresis of a 5 uL aliquot on a 1% agarose gel, and comparison ofthe product band to a mass ladder.

Excess primers, nucleotides, enzyme and template were removed from thePCR product using the MoBio PCR clean up Kit. The cleaned PCR productwas subjected to direct sequencing using the primers described above.

The forward (27F, SEQ ID NO:1) and reverse sequences (1525R, SEQ IDNO:2) were aligned using the BioEdit software, and a 1451 by consensussequence was created (see SEQ ID NO:3, infra). The 16S rRNA geneconsensus sequence of strain H491 was compared to availablerepresentative bacterial 16S sequences using BLAST. For strain H491, theclosest match was to Bacillus megaterium (GenBank accession numberCP001983.1), with 99% similarity.

27F Sequence (SEQ ID NO: 1):NNNNNNNNNGNNNGCTATAATGCAAGTCGAGCGAACTGATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACAAGAGTAACTGCTTGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACNGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCNCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTNNGATAGAGCGTTCCCCTTNNGGGACAGAGTGACAGGTGGNGCATGGGTTGTCGTCAGCTCNTGTCGTGAGATNNTGGGTTAAGTCCCGCAACGAGCGCAACCNTTGATCTANNNCAGCATTCANNNGGNANTCTNNNNGACTGCNGNTGANNACCGNAGAAAGN TGGGGATGACNN1525R Sequence (SEQ ID NO: 2):NNNNNNNNNNNNNNNCGACTTCaCCCCAATCATCTGTCCCACCTTAGGCGGCTAGCTCCTTACGGTTACTCCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTATGGGATTGGCTTGACCTCGCGGTCTTGCAGCCCTTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCCGAAGGGGAACGCTCTATCTCTAGAGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAAAAAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCGCTTTTCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCANACTTAANAAACCGCCTGCGCGCGCTTTACGCCCAATAATTNCNGATAACGCTTGNCACCTACGTATTACCGCGGCTGCTGGCACGTANNTAGCCGNGGNTTTCTGGTTAGGTACNGTCNNGGTACAAGCANNTACTCTNNACTNNNNTTNCNTAACANANANTTTACGACCCGAANNCNTCNTCACTCANGCGCNTNGCTCNNNCAGANTNTNNNNN Consensus Sequence (SEQ ID NO: 3):ATAATGCAAGTCGAGCGAACTGATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACAAGAGTAACTGCTTGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACNGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCTAGCCGCCTAAGGTGGGACAGATGATTGGGGT GFor strain M018, the closest match was found to be to Bacillusmegaterium strain PSB55 (GenBank Accession number HQ242768), with a 99%Match. Identification was performed as described for strain H491 above.

M018 FD1 Sequence (SEQ ID NO: 4)GCTATAcTGCAAGTCGAGCGAACTGATAGAAGCTTGCTTCTATGACGTTGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACAAGAGTAACTGCTTGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTGATCTTAGTTGCCAGCATTTAGTGGGCACTCTAAGGTGACTGCCGGTGACAACGAGAAGGTGGGGATGACGTCGAATCATCATGCCCCTTAKGACCTGGGGCTWCACAMCGKTGCYWACAAK GGAATTGGTTACM018 RD1 Sequence (SEQ ID NO: 5):TGTTACGACTTCACCCCAATCATCTGTCCCACCTTAGGCGGCTAGCTCCTTACGGTTACTCCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACYAGCGATTCCTGCTTCATGTAGGCKAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTATGGGATTGGCTTGACCTCGCGGTCTTGCAGCCCTTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTAAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACMACCATGCACCACCTGTCACTCTGTCCCCCGAAGGGGAACGCTCTATCTCTAGAGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAAAAAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAAATCCGCTTTTCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCGCGCTTTACGCCCAATAATTCAGATAACGCTCGCCACCTACGTATTACCGCGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGTACCGTCAGTACAGCAGTACTCTGTACTTGTTCTTCCTAACAACAGAGTTTACGACCCGAAAGCCTTCATCATTC M018 Consensus Sequence(SEQ ID NO: 6) GCTATACTGCAAGTCGAGCGAACTGATAGAAGCTTGCTTCTATGACGTTGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACAAGAGTAACTGCTTGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCRAGCGTTATCYGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAWTTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTKGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTMGCCTACATGAAGCAGGAATCGCTRGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCTAGCCGCCTAAGGTGGGACAGATGATTGGGGT GAAGTCGTAACAFor strain J142, the closest match was to Bacillus megaterium strainZFJ-14. Identification was performed as described for strain H491 above.

J142 FD1 Sequence (SEQ ID NO: 7):TGCTATAATGCAGTCGAGCGAACTGATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGGGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCMACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACRAGAGTAACTGCTTGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAGAACCTTACCAGGTCTTGACATCCTCTGACACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGTGTGCATGGTGTCGTCAGCTCGTGTCGTGAGATGTGGGTAGTCCCGCACGAGCGCACCTGATCTAGTGCAGCATTAGTGGCACTCTAGTGACTGCGTGACACGAGGAGGTGGGATGACGTCATCATCATGCCCCTATGACTGGGCTACCACGTGCTACATGGATGTCAAGGCTGCAGACCGAAGTCAGCAATCATAAACATTCTCAGTCGAATGTAAGTCA J142 RD1 Sequence (SEQ ID NO: 8):CTTGTTCGACTTCCCCCAATCATCTGTCCCACCTTAGGCGGCTAGCTCCTTACGGTTACTCCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTATGGGATTGGCTTGACCTCGCGGTCTTGCAGCCCTTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTAAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCCGAAGGGGAACGCTCTATCTCTAGAGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAAAAAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCGCTTTTCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCGCGCTTTACGCCCAATAATTCCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCGAGGTACAAGCAGTTACTCTCGTACTTGTCTTCCCTAACACAGAGTTTTACGACCCGAAGCTCATCACTCAGCGCGTGCTCGTCGACTTCGTCATTGCGAGATCCCTACTGCTGCTTCCGTAGGAGTCTGGACCTGTCTCAGTCAGGTGTGACGGATCACCCTCTTCAGTCGCCTATGTGCCACTCTCGTGGGTCCCG J142 Consensus Sequence (SEQ ID NO: 9)TGCTATAATGCAGTCGAGCGAACTGATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGGGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCMACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACGAGAGTAACTGCTTGTACCTYGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCTAGCCGCCTAAGGTGGGACAGATGATTG GGGGAAGTCGAACAAG

recA Sequences

PCR reactions for the amplification of the recA gene were set up bycombining 2 μL of the clean DNA extract with 25 μL of GoTaq GreenMastermix, 1.5 μL forward primer (recAf, 5′-GATCGTCARGCAGSCYTWGAT-3′,SEQ ID NO:12), and 1.5 μL reverse primer (recAr,5′-TTWCCRACCATAACSCCRAC-3′, SEQ ID NO:13). The reaction volume wasbrought up to 50 μL using sterile nuclease-free water. The PCR reactionwas conducted on a thermocycler machine under the following conditions:5 minutes at 95° C. (initial denaturing), 30 cycles of 30 seconds at 95°C., 30 seconds at 45° C. and 1 minute at 72° C., followed by 5 minutesat 72° C. (final extension) and a final hold temperature of 4° C.

The size, quality and quantity of the PCR product was evaluated byrunning a 5 μL aliquot on a 1% agarose gel, and comparing the productband to a mass ladder.

Excess primers, nucleotides, enzyme and template were removed from thePCR product using the MoBio PCR clean up Kit. The cleaned PCR productwas subjected to direct sequencing using the primers described above.

The forward and reverse sequences were aligned using the BioEditsoftware, and a 505 by consensus sequence was created.

The recA gene consensus sequence of each strain (H491, J142 and M018)was compared to representative bacterial sequences using BLAST. Theclosest species match was to the complete genome of Bacillus megaterium(accession number CP001982.1) with 99% similarity.

H491 recA Forward Sequence (SEQ ID NO: 14):    TGAAAGCATTTGGTAAGGTTCAATTATGAAATTAGGTGAACAAACGGAAAAAAGAATTTCTACAATTCCAAGTGGTTCATTGGCGTTAGATATAGCCTTAGGTGTAGGTGGATATCCACGTGGACGTGTAGTTGAAGTATATGGTCCAGAAAGCTCAGGTAAAACAACAGTTGCTCTTCATGCGATTGCAGAAGTTCAACAGCAGGGCGGACAGGCTGCATTTATCGATGCGGAGCACGCGTTAGATCCTGTATATGCTCAAAAATTAGGTGTGAATATTGATGAGCTATTATTATCTCAGCCTGATACGGGAGAACAAGCTTTAGAAATCGCTGAAGCTTTAGTTCGAAGCGGTGCAGTAGATATTATCGTTGTTGACTCAGTAGCAGCATTAGTGCCAAAAGCGGAAATTGAAGGAGAAATGGGAGACTCTCACGTGGGTCTACAAGCTCGTTTAATGTCTCAAGCATTGCGTAAACTATCTGGAGCTATCAATAAGTCTAAAACAATCGCTATCTTTATTAACCAAATTCGTGAAAAAGTCGG CGTTnGGGTCGGAAAAH491 recA reverse Sequence (SEQ ID NO: 15):    GWAGCGATTGTTTTAGACTTATTGATAGCTCCAGATAGTTTACGCAATGCTTGAGACATTAAACKAGCTTGTAGACCCACGTGAGAGTCTCCCATTTCTCCTTCAATTTCCGCTTTTGGCACTAATGCTGCTACTGAGTCAACAACGATAATATCTACTGCACCGCTTCGAACTAAAGCTTCAGCGATTTCTAAAGCTTGTTCTCCCGTATCAGGCTGAGATAATAATAGCTCATCAATATTCACACCTAATTTTTGAGCATATACAGGATCTAACGCGTGCTCCGCATCGATAAATGCAGCCTGTCCGCCCTGCTGTTGAACTTCTGCAATCGCATGAAGAGCAACTGTTGTTTTACCTGAGCTTTCTGGACCATATACTTCAACTACACGTCCACGTGGATATCCACCTACACCTAAGGCTATATCTAACGCCAATGAACCACTTGGAATTGTAGAAATTCTTTTTTCCGTTTGTTCACCTAATTTCATAATTGAACCTTTACCAAATTGCTTTTCAATTTGTTTTAAAGCCATATCWAAGCCT GCWWWGACGATCH491 Consensus Sequence (SEQ ID NO: 16):    AAGGTTCAATTATGAAATTAGGTGAACAAACGGAAAAAAGAATTTCTACAATTCCAAGTGGTTCATTGGCGTTAGATATAGCCTTAGGTGTAGGTGGATATCCACGTGGACGTGTAGTTGAAGTATATGGTCCAGAAAGCTCAGGTAAAACAACAGTTGCTCTTCATGCGATTGCAGAAGTTCAACAGCAGGGCGGACAGGCTGCATTTATCGATGCGGAGCACGCGTTAGATCCTGTATATGCTCAAAAATTAGGTGTGAATATTGATGAGCTATTATTATCTCAGCCTGATACGGGAGAACAAGCTTTAGAAATCGCTGAAGCTTTAGTTCGAAGCGGTGCAGTAGATATTATCGTTGTTGACTCAGTAGCAGCATTAGTGCCAAAAGCGGAAATTGAAGGAGAAATGGGAGACTCTCACGTGGGTCTACAAGCTCGTTTAATGTCTCAAGCATTGCGTAAACTATCTGGAGCTATCAATAAGTCTAAAACAAT CGCT

M018: recA Gene Sequencing

M018 recA forward Sequence (SEQ ID NO: 17):    TTGAAGCATTTGGTAAAGGTTCAATTATGAAATTAGGTGAACAAACGGAAAAAAGAATTTCTACAATTCCAAGTGGTTCATTAGCGTTAGATATAGCTTTAGGTGTAGGTGGATATCCACGTGGACGCGTAGTTGAAGTATATGGTCCAGAAAGCTCAGGTAAAACAACAGTTGCTCTTCATGCGATTGCAGAAGTTCAACAGCAGGGCGGACAGGCTGCATTTATCGATGCGGAGCACGCGTTAGATCCTGTATATGCTCAAAAATTAGGTGTGAATATTGATGAGCTATTATTATCTCAGCCTGATACGGGAGAACAAGCTTTAGAAATCGCTGAAGCTTTAGTTCGAAGCGGTGCAGTAGATATTATCGTTGTTGACTCAGTAGCAGCATTAGTGCCAAAAGCGGAAATTGAAGGAGAAATGGGAGACTCTCACGTGGGTCTACAAGCTCGTTTAATGTCTCAAGCATTGCGTAAACTATCTGGAGCTATCAACAAGTCTAAAACAATCGCTATCTTTATTAACCAAATTCGTGAAAAAGTCG GCGTTnGGGTTCGGAAAAM018 recA reverse Sequence (SEQ ID NO: 18):    GGTATAAAGATAGGCGATTGTTTTAGACTTGTTGATAGCTCCAGATAGTTTACGCAATGCTTGAGACATTAAAcgAGCTTGTAGACCCACGTGAGAGTCTCCCATTTCTCCTTCAATTTCCGCTTTTGGCACTAATGCTGCTACTGAGTCAACAACGATAATATCTACTGCACCGCTTCGAACTAAAGCTTCAGCGATTTCTAAAGCTTGTTCTCCCGTATCAGGCTGAGATAATAATAGCTCATCAATATTCACACCTAATTTTTGAGCATATACAGGATCTAACGCGTGCTCCGCATCGATAAATGCAGCCTGTCCGCCCTGCTGTTGAACTTCTGCAATCGCATGAAGAGCAACTGTTGTTTTACCTGAGCTTTCTGGACCATATACTTCAACTACGCGTCCACGTGGATATCCACCTACACCTAAAGCTATATCTAACGCTAATGAACCACTTGGAATTGTAGAAATTCTTTTTTCCGTTTGTTCACCTAATTTCATAATTGAACCTTTACCAAATTGCTTTTCAATTTGTTTTAAAGCCATATCWAAGCCTAAWWRGACGATCYA Consensus Sequence (SEQ ID NO: 19):    AAGGTTCAATTATGAAATTAGGTGAACAAACGGAAAAAAGAATTTCTACAATTCCAAGTGGTTCATTAGCGTTAGATATAGCTTTAGGTGTAGGTGGATATCCACGTGGACGCGTAGTTGAAGTATATGGTCCAGAAAGCTCAGGTAAAACAACAGTTGCTCTTCATGCGATTGCAGAAGTTCAACAGCAGGGCGGACAGGCTGCATTTATCGATGCGGAGCACGCGTTAGATCCTGTATATGCTCAAAAATTAGGTGTGAATATTGATGAGCTATTATTATCTCAGCCTGATACGGGAGAACAAGCTTTAGAAATCGCTGAAGCTTTAGTTCGAAGCGGTGCAGTAGATATTATCGTTGTTGACTCAGTAGCAGCATTAGTGCCAAAAGCGGAAATTGAAGGAGAAATGGGAGACTCTCACGTGGGTCTACAAGCTCGTTTAATGTCTCAAGCATTGCGTAAACTATCTGGAGCTATCAACAAGTCTAAAACAAT CG

J142: recA Gene Sequencing

J142 recA Forward Sequence (SEQ ID NO: 20):    GAAAGCATTTGGTAAGGTTCAATTATGAAATTAGGTGAACAAACGGAAAAAAGAATTTCTACAATTCCAAGTGGTTCATTAGCGTTAGATATAGCCTTAGGTGTAGGTGGATATCCACGTGGACGTGTAGTTGAAGTATATGGTCCAGAAAGCTCAGGTAAAACAACAGTTGCTCTTCATGCGATTGCAGAAGTTCAACAGCAGGGCGGACAGGCTGCATTTATCGATGCGGAGCACGCGTTAGATCCTGTATATGCTCAAAAATTAGGTGTGAATATTGATGAGCTATTATTATCTCAGCCTGATACGGGAGAACAAGCTTTAGAAATCGCTGAAGCTTTAGTTCGAAGCGGTGCAGTAGATATTATCGTTGTTGACTCAGTAGCAGCATTAGTGCCAAAAGCGGAAATTGAAGGAGAAATGGGAGACTCTCACGTGGGTCTACAAGCTCGTTTAATGTCTCAAGCATTGCGTAAACTATCTGGAGCTATCAATAAGTCTAAAACAATCGCTATCTTTATTAACCAAATTCGTGAAAAAGTCGGC GTTnGGGTCGGAAAAJ142 recA Reverse Sequence (SEQ ID NO: 21):    AGCGATTGTTTTAGACTTATTGATAGCTCCAGATAGTTTACGCAATGCTTGAGACATTAAACGAGCTTGTAGACCCACGTGAGAGTCTCCCATTTCTCCTTCAATTTCCGCTTTTGGCACTAATGCTGCTACTGAGTCAACAACGATAATATCTACTGCACCGCTTCGAACTAAAGCTTCAGCGATTTCTAAAGCTTGTTCTCCCGTATCAGGCTGAGATAATAATAGCTCATCAATATTCACACCTAATTTTTGAGCATATACAGGATCTAACGCGTGCTCCGCATCGATAAATGCAGCCTGTCCGCCCTGCTGTTGAACTTCTGCAATCGCATGAAGAGCAACTGTTGTTTTACCTGAGCTTTCTGGACCATATACTTCAACTACACGTCCACGTGGATATCCACCTACACCTAAGGCTATATCTAACGCTAATGAACCACTTGGAATTGTAGAAATTCTTTTTTCCGTTTGTTCACCTAATTTCATAATTGAACCTTTACCAAATTGCTTTTCAATTTGTTTTAAAGCCATATCAAAGCCTGC TRAACRATCAAConsensus Sequence (SEQ ID NO: 22):    AAGGTTCAATTATGAAATTAGGTGAACAAACGGAAAAAAGAATTTCTACAATTCCAAGTGGTTCATTAGCGTTAGATATAGCCTTAGGTGTAGGTGGATATCCACGTGGACGTGTAGTTGAAGTATATGGTCCAGAAAGCTCAGGTAAAACAACAGTTGCTCTTCATGCGATTGCAGAAGTTCAACAGCAGGGCGGACAGGCTGCATTTATCGATGCGGAGCACGCGTTAGATCCTGTATATGCTCAAAAATTAGGTGTGAATATTGATGAGCTATTATTATCTCAGCCTGATACGGGAGAACAAGCTTTAGAAATCGCTGAAGCTTTAGTTCGAAGCGGTGCAGTAGATATTATCGTTGTTGACTCAGTAGCAGCATTAGTGCCAAAAGCGGAAATTGAAGGAGAAATGGGAGACTCTCACGTGGGTCTACAAGCTCGTTTAATGTCTCAAGCATTGCGTAAACTATCTGGAGCTATCAATAAGTCTAAAACAAT CGCT

All isolates were 99-100% match to Bacillus megaterium DSM319, as wellas other strains of Bacillus megaterium. These results confirm theidentity of the three isolates as Bacillus megaterium.

Example 3 Biochemical Characterization of B. Megaterium H491

Bacillus megaterium strain H491 was determined to be gram positive,urease positive, catalase positive, oxidase negative and lipasepositive. Extensive biochemical characterization of the isolate wasperformed using Biolog Phenotypic Microarrays. The full BiologPhenotypic microarray consists of twenty 96-well plates, each wellcontaining a different carbon, nitrogen, phosphorus or other nutrientsource. Some of the plates also contain different antibiotics, metals,etc., to evaluate susceptibility of the isolate. Absorbance readingsfrom the wells in each plate were compared to a negative control well.Absorbance readings above the negative control threshold indicatedgrowth of the isolate under the conditions in a particular well, whilereading at or below the negative control indicated that the isolatefailed to thrive under the specific conditions of that well. The testwas performed for H491 at 25° C., with duplicate plates for eachphenotype test.

B. megaterium strain H491 was able to utilize the followingcarbohydrates, organic acids and peptides as carbon sources:L-Arabinose, N-Acetyl-D-Glucosamine, D-Galactose, L-Aspartic acid,L-Proline, D-Trehalose, Dulcitol, Glycerol, D-Glucuronic acid,D-Gluconic acid, D-Xylose, L-Lactic acid, D-Mannitol, L-Glutamic acid,D-Glucose-6-Phosphate, D-Galactonic acid-g-Lactone, D,L-Malic acid,D-Ribose, D-Fructose, a-D-Glucose, Maltose, D-Melibiose, Thymidine,L-Asparagine, D-Aspartic acid, a-Ketoglutaric acid, Sucrose,L-Glutamine, Maltotriose, Citric acid, Fumaric acid, Bromosuccinic acid,L-Alanine, Ala-Gly, Methylpyruvate, L-Malic acid, L-Lyxose, Pyruvicacid, Dextrin, Glycogen, D-Arabinose, Arbutin, 2-Deoxy-D-Ribose,3-O-b-D-Galactopyranosyl-D-Arabinose, Gentiobiose, Palatinose,D-Raffinose, Salicin, Stachyose, D-Tagatose, Turanose, g-Amino-N-Butyricacid, D-Glucosamine, b-Hydroxybutyric acid, 5-Keto-D-Gluconic acid,Quinic acid, D-Tartaric acid, L-Tartaric acid, L-Ornithine,L-Pyroglutamic acid, and Dihydroxyacetone.

The following substrates were not utilized as carbon sources, asevidenced by absorbance readings below the negative control threshold:D-Saccharic acid, D-Serine, D-Sorbitol, L-Fucose, D,L-a-GlycerolPhosphate, L-Rhamnose, D-Glucosaminic acid, 1,2-Propanediol,a-Ketobutyric acid, a-D-Lactose, D-Glucose-1-Phosphate,a-Hydroxyglutaric acid-g-Lactone, a-Hydroxybutyric acid, Adonitol,2′-Deoxyadenosine, Adenosine, D-Threonine, Propionic acid, Mucic acid,Glycolic acid, Tricarballylic acid, L-Threonine, N-Acetyl-D-Mannosamine,D-Malic acid, Gly-Pro, L-Galactonic acid-g-Lactone, Chondroitin SulfateC, a-Cyclodextrin, Inulin, Laminarin, Mannan, N-Acetyl-D-Galactosamine,N-Acetyl-Neuraminic acid, b-D-Allose, Amygdalin, D-Arabitol, L-Arabitol,i-Erythritol, D-Fucose, L-Glucose, D-Lactitol, b-Methyl-D-Galactoside,3-Methylglucose, b-Methyl-D-Glucuronic acid, a-Methyl-D-Mannoside,Sedoheptulosan, L-Sorbose, Xylitol, N-Acetyl-D-Glucosaminitol, d-AminoValeric acid, Capric acid, Caproic acid, Citraconic acid, Citramalicacid, 2-Hydroxybenzoic acid, 4-Hydroxybenzoic acid, a-Keto-Valeric acid,Itaconic acid, D-Lactic acid Methyl Ester, Malonic acid, Melibionicacid, Oxalic acid, D-Ribono-1,4-Lactone, Sebacic acid, Sorbic acid,Succinamic acid, Acetamide, L-Alaninamide, N-Acetyl-L-Glutamic acid,Glycine, L-Homoserine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine,L-Lysine, L-Phenylalanine, L-Valine, D,L-Carnitine, sec-Butylamine,D,L-Octopamine, 2,3-Butanediol, 2,3-Butanedione, and3-Hydroxy-2-butanone.

H491 was able to utilize the following amino acids as nitrogen sources:L-Glutamine, L-Pyroglutamic acid, L-Glutamic acid, L-Asparagine,L-Ornithine, D-Asparagine, L-Proline, L-Aspartic acid, D-Alanine,L-Arginine, L-Alanine, L-Serine, D-Aspartic acid, L-Tryptophan, andL-Tyrosine. It was also able to utilize Urea, Putrescine, Agmatine,N-Acetyl-D-Glucosamine, Cytidine, Guanosine, Inosine, Xanthine, Uricacid, Allantoin, g-Amino-N-Butyric acid and a-Amino-N-Valeric acid asadditional organic nitrogen sources. H491 could also utilize ammonia,nitrite and nitrate as inorganic nitrogen sources.

H491 was able to utilize a variety of substrates as phosphorous sources,including Phosphate, Thiophosphate, Dithiophosphate,D-Glucosamine-6-Phosphate, Cysteamine-S-Phosphate, Uridine 2′,3′-CyclicMonophosphate, and Thymidine 5′-Monophosphate.

Metabolism of H491 was inhibited below pH 5, but restored at pH 4.5 inthe presence of L-arginine, L-methionine and 5-hydroxy-lysine. Growthwas observed between pH 5 to pH 10. The strain did not tolerate NaClabove 5%, and only slight growth was detected at 4% NaCl. The Bergey'sManual of Systematic Bacteriology indicates that Bacillus megateriumisolates can use citrate as a sole carbon source; most can grow at 7%NaCl, but none at 10% NaCl; and most strain do not reduce nitrate. Incontrast, H491 did not tolerate NaCl above 5%, and was able to utilizeboth nitrate and nitrite as nitrogen sources.

Example 4 Production of B. Megaterium H491, M018 and J142 byFermentation

A supernatant with nematicidal activity was produced through thesubmerged fermentation of strain H491 under aerobic conditions in liquidV8 medium. Other suitable media include tryptic soy broth, or anynutrient medium containing appropriate carbon and nitrogen sources.

A seed plate was started by streaking a fresh potato dextrose agar platewith a small amount of strain H491, using a sterile loop. The plate wasincubated at 25° C. for 2-3 days or until enough biomass was evident onthe surface of the plate.

A 50 mL V8 medium seed flask was inoculated with one loopful of materialcollected from the agar plate surface. The seed was incubated in ashaker at 200 rpm for 2 days.

A glass 2.8 L, non-baffled fernbach flask containing 500 mL of V8 mediumwas aseptically inoculated with 2% of seed. The fermentation was allowedto proceed at 25° C. for 5 days with constant agitation at 150-200 rpm.

The supernatant was obtained by separation the cells from the spentfermentation broth by centrifugation, or other means of separation.Activity of the supernatant was verified by means of the bioassaydescribed below.

Example 5 Further Characterization of B. Megaterium H491, J142 and M018Resistance to Antibiotics

Antibiotic susceptibility of Bacillus megaterium strains H491, J142 andM018 was tested using antibiotic disks on Muller-Hinton medium asdescribed in PML Microbiological's technical data sheet #535. Resultsobtained after 48-hour incubation at 25° C. are presented in Table 1.

TABLE 1 Susceptibility of Bacillus megaterium H491, J142 and M018 tovarious antibiotics. Susceptibility degree is indicated by +++ (very),++ (somewhat), + (marginally), and resistance is indicated by (−).Concentration (mg) H491 J142 M018 Tetracycline 30 +++ +++ +++ Kanamycin30 +++ +++ +++ Erythromycin 15 +++ +++ +++ Streptomycin 10 +++ +++ +Penicillin 10 +++ − ++ Ampicillin 10 +++ +++ ++ Oxytetracycline 30 ++++++ +++ Chloramphenicol 30 +++ +++ ++ Ciprofloxacin 5 +++ +++ +++Gentamicin 10 +++ +++ +++ Piperacillin 100 +++ + ++ Cefuroxime 30 ++++++ +++ Imipenem 10 +++ +++ +++ Sulphamethoxazole- 23.75/25 +++ +++ +++Trimethoprim

Chemical Sensitivity

Chemical sensitivity data was obtained from the Biolog PhenotypicMicroarray. H491 was found to be susceptible to the following compounds:Chlortetracycline, Lincomycin, Amoxicillin, Cloxacillin, Minocycline,Capreomycin, Demeclocycline, Nafcillin, Cefazolin, Enoxacin, Nalidixicacid, Chloramphenicol, Erythromycin, Neomycin, Cephalothin, Kanamycin,Penicillin G, Tetracycline, Carbenicillin, Oxacillin, Penimepicycline,Paromomycin, Vancomycin, Sisomicin, Novobiocin,2,4-Diamino-6,7-diisopropylpteridine, Sulfadiazine, BenzethoniumChloride, Tobramycin, Sulfathiazole, 5-Fluoroorotic acid,Sulfamethoxazole, L-Aspartic-b-hydroxamate, Spiramycin, Rifampicin,Dodecyltrimethyl ammonium bromide, Azlocillin, 2,2′-Dipyridyl,6-Mercaptopurine monohydrate, Doxycycline, Potassium chromate,Cefuroxime, 5-Fluorouracil, Rolitetracycline, Cesium chloride, Thallium(I) acetate, Cobalt (II) chloride, Trifluoperazine, Tylosin,Acriflavine, Furaltadone, Sanguinarine chloride, Fusaric acid, Boricacid, 1-Hydroxypyridine-2-thione (pyrithione), Sodium Cyanate, Cadmiumchloride, Iodoacetic acid, Sodium Dichromate, Cefoxitin, Sodiummetaborate, Chloramphenicol, Sodium metavanadate, Chelerythrinechloride, Carbenicillin, Sodium Nitrite, EthyleneGlycol-bis(b-Aminoethyl ether)-N,N,N′,N′-Tetraacetic Acid, Promethazine,Sodium orthovanadate, Guanidine hydrochloride, D-Cycloserine, EDTA,5,7-Dichloro-8-hydroxyquinaldine, 5,7-Dichloro-8-hydroxyquinoline,Fusidic acid, sodium salt, 1,10-Phenanthroline Monohydrate, Phleomycin,Domiphen bromide, Alexidine, 5-Nitro-2-furaldehyde semicarbazone(Nitrofurazone), Methyl viologen, Oleandomycin, phosphate salt,Puromycin, Carbonyl-cyanide m-chlorophenylhydrazone (CCCP), SodiumAzide, Menadione, sodium bisulfite, 2-Nitroimidazole, Hydroxyurea,5-Chloro-7-iodo-8-hydroxy-quinoline, Sulfanilamide, Trimethoprim,Dichlofluanid, Protamine sulfate, Chlorodinitrobenzene, Diamide,Cinoxacin, Streptomycin, Rifamycin SV, Potassium tellurite, SodiumSelenite, Glycine hydroxamate, 4-Chloro-3,5-dimethyl-phenol, D-Serine,Thiosalicylate, Salicylate, sodium, Sulfachloropyridazine, Oxycarboxin,3-Amino-1,2,4-triazole, Chlorpromazine, Niaproof, Compound 48/80, SodiumTungstate, Lithium chloride, Chlorambucil, Cefamandole nafate,Cefsulodin, Caffeine, Ketoprofen, Thiamphenicol, Trifluorothymidine,Poly-L-lysine, Pentachlorophenol, Sodium Arsenite, Lidocaine, Sodiumperiodate, Antimony (III) chloride, Semicarbazide hydrochloride,Tinidazole, 5-Fluoro-5′-deoxyuridine, 2-Phenylphenol, Plumbagin,Josamycin, Gallic acid, Methyltrioctylammonium chloride,2,4-Dintrophenol, Chlorhexidine diacetate, trans-Cinnamic acid,Tetraethylthiuram disulfide, FCCP, D,L-Thioctic acid, Phenethicilllin,Sodium Caprylate, Lauryl sulfobetaine(N-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Hexamine cobalt(III) chloride, Polymyxin B, Amitriptyline, Apramycin, Orphenadrine,D,L-Propanolol, Tetrazolium violet, Thioridazine, Atropine, Ornidazole,Proflavine, 18-Crown-6 ether, Crystal violet, Dodine(n-Dodecylguanidine), Hexachlorophene, 4-Hydroxycoumarin,Oxytetracycline, Pridinol, Captan, 3,5-Dinitrobenzoic acid,8-Hydroxyquinoline, Patulin, Tolylfluanid, and Troleandomycin.

Resistance was observed in wells containing the following compounds withknown antibacterial activity (at a maximum concentration of 4 ug):amikacin, lomefloxacin, bleomycin, colistin, gentamicin, ofloxacin,polymixin B, sulfamethazine, spectinomycin, ampicillin and oxolinicacid.

Example 6 Fatty Acid Composition

After incubation for 24 hours at 28° C., a loopful of well-grown cellswere harvested and fatty acid methyl esters were prepared, separated andidentified using the Sherlock Microbial Identification System (MIDI) asdescribed (see Vandamme et al., 1992). The predominant fatty acidspresent in the Bacillus megaterium strains are shown in Table 2.

TABLE 2 Fatty Acid Methyl Ester (FAME) composition (%) of the differentBacillus megaterium strains with nematicidal activity. FAME M018 J142H491 14:0i 4.77 6.05 6.36 14:0 1.33 1.65 1.52 15:0i 41.89 32.97 32.0315:0ai 36.7 42.93 44.41 16:01ω7cOH 1.32 1.39 1.03 16:0i 0.97 1.41 2.1116:1ω11c 3.56 4.03 3.16 16:0 2.06 2.96 3.47 17:1iω10c 1.38 0.56 1.95 Sumin 4 1.08 0.71 3.97 17:0i 2.22 1.89 1.95 17:0ai 2.73 3.43 3.97Similarity 0.949 0.948 0.991 index to B. megaterium

Similarity indices of the FAME profiles to the database were all withinthe species confidence threshold (0.948-0.991). A dendogram was built bycluster analysis techniques to produce unweighted pair matching based onfatty acid compositions. The results indicate that H491 (listed asMBI-303) is most similar to J142, and that M018 is more different fromthe other two strains.

Example 7 MALDI-TOF Identification of Isolates H491, M018 and J142

Samples were submitted for MALDI-TOF (Matrix Assisted LaserDesorption/Ionization-Time of Flight) spectrometry profiling andidentification to MIDI Labs, Inc (Newark, Del.) to create a profile fromthe ribosomal proteins of the isolates. The profile was compared to aproprietary database, and the isolates were identified based on theirMALDI-TOF scores.

MALDI-TOF scores above 2.000 provide identification confidence to thespecies level, scores from 1.700-1.999 to the genus level, and scoresbelow 1.700 indicate no match in the database. Both H491 and M018yielded MALDI-TOF scores above 2.000 (2.163 and 2.291 respectively),while J142 had a score of 1.972. These results indicate that theribosomal protein profile of J142 may be unique within the Bacillusmegaterium species.

Example 8 Efficacy of Bacillus megaterium Isolates Against Root-KnotNematodes (Meloidogyne incognita)-Agar Assays A: Water-Agar-Assay#1

To determine and compare the effects of different Bacillus megateriumstrains on root-knot nematodes (Meloidogyne incognita VW6), awater-agar-assay was conducted. Seven-day-old ‘White Wonder’ cucumberseedlings were transferred on water agar in 60-mm-diameter petri dishesat a rate of one seeding/plate. Cucumbers were then treated with 300 mlof B. megaterium H491 fermentation supernatant and 700 M.incognita/plate, with six replications of each treatment. Afterincubation for 11 days, the number of galls in cucumber roots wasrecorded. The results, shown in Table 3, indicate that all strainssignificantly reduced the number of galls in cucumber roots comparedwith the water control.

TABLE 3 Effects of Bacillus megaterium isolates' fermentationsupernatant on root-knot gall formation in cucumber roots Treatment GallNumber % Gall Reduction H491 3.3 ± 1.4 66.7 ± 13.7 J142 3.7 ± 1.2 63.3 ±12.1 M018 5.8 ± 1.5 41.7 ± 14.7 Avid (1%)   1 ± 0.5  95 ± 5.5 MediaBlank   7 ± 1.2 33.3 ± 12.1 Water  10 ± 2.6  0 ± 26

B: Agar Seedling Assay #2: Effect of B. Megaterium H491 on M. IncognitaInfestation on Cucumber

To assess the effect of the top supernatant of B. megaterium H491 onjuveniles (J2s) of root-knot nematodes (Meloidogyne incognita VW6), thefollowing test was conducted on 60 mm-diameter petri dishes:

Seeds of cucumber cv. ‘White Wonder’ were germinated on wet tissue paperin petri dishes at room temperature. A week later, germinated seedlingswere transferred to water agar in petri dishes at the rate of oneseedling/dish. A 300-ml aliquot of B. megaterium. H491 fermentationsupernatant was added into each dish, after which 300 M. incognita J2swere added in 150 ml of deionized water. Petri dishes were then coveredand incubated at 25° C. for 7 days. Water, media blank and Avid (0.1%)were used as negative, negative, and positive controls, respectively.The effect of each substance on nematode survival was assessed after 7days by counting the number of galls in cucumber roots, and expressed asa percentage of the water control. Experiments were conducted induplicate.

The results, presented in FIG. 9, show that B. megaterium H491supernatant significantly reduced the number of galls in cucumber rootscompared with the water control in both trials, which indicated thatH491 was effective against M. incognita and can be used for managementof this nematode in cucumber.

Example 9 In Vitro Assays with M. Incognita and M. Hapla A: In VitroNematicidal Bioassay Against M. Incognita and M. Hapla

In order to ensure consistent and adequate control of nematodes in allfilter sterilized B. megaterium H491 fermentations, the motility ofroot-knot nematode juveniles (J2's) was measured in an in vitro 96-wellplastic cell-culture plate bioassay. This procedure is based on a visualgrading of motility of the nematodes in each well treated with H491where each treatment is tested in well repetitions of four: 100 ml ofeach microbial sample solution was dispensed into its corresponding wellfollowed by 30 ml of a 1:100 dilution of plant preservative material(PPM) solution that momentarily suppresses microbial growth and allowsvisibility in each well. Finally, 50 ml of nematode solution, containingapproximately 15 J2's, was added. The plate was covered and incubatedfor 24 hours at 25° C., then the effect of each sample was visuallyscored and recorded. Nematicidal activity was confirmed in 19 differentsmall scale fermentations, with nematicidal activity usually hoveringover 60%, as summarized in FIG. 5.

B: Nematode (M. Hapla) Recovery after Exposure to H491 Supernatant

To assess the effect of H491 supernatant on the motility and subsequentrecovery of juveniles of root-knot nematodes (Meloidogyne hapla), thefollowing test was conducted on 96-well plastic cell-culture plates. A50-ml aliquot of each test solution was added into appropriate wellsafter which, 50 J2s dispensed in 30 ml of deionized water were addedinto each well. The plate was closed with a lid, and incubated at 25° C.for 72 hours. Water and Avid® nematicide (1%) were used as negative andpositive controls, respectively. The effect of each substance onnematode motility was determined after 24, 48, and 72 hours by adding adrop of 1 N NaOH into each well, and the proportion of motile nematodesin each treatment was recorded as a percentage of the initial number. Toassess the recovery of motility in each treatment, a volume of 70 ml wasremoved from each well, and the remaining solution in each well wasdiluted by adding 100 ml of deionized water. Plates were again incubatedfor 24 hours as described above, after which the second motilityevaluation was performed. There were three replications for eachtreatment and the study was conducted twice.

Results are shown in Table 4. After 48 hours, wells containing nematodesexposed to the highest amount of treatment solution were too cloudy toread without washing off the treatments. Therefore, percent of motilenematodes were not recorded directly after 48 or 72 hours. Percent ofmotile nematodes exposed to H491 supernatant was found to decrease withincreased time of incubation. The lowest rate (17.5%) was observed 72hours after incubation. These results indicate that H491 supernatant canimmobilize juveniles of root-knot nematodes, and this effect can lastfor at least 72 hours.

TABLE 4 Percent recovery rate of Meloidogyne hapla after 24, 48, and 72hours of incubation with H491 supernatant. Treatment* Candidate 24 24-2448-24 72-24 Water 95 ± 3.2 97.5 ± 2.7   95 ± 4.5 90 ± 4.5 Media 78.3 ±2.6   82.5 ± 2.7 82.5 ± 2.7 80 ± 4.5 Avid (1%) 10 ± 4.5 13.3 ± 2.6 11.7± 2.6 10 ± 4.5 H491 38 ± 2.7   35 ± 4.5   25 ± 5.5 17.5 ± 2.7   Datashown are mean values obtained from two studies. *“24”indicates thatobservation was conducted directly 24 h after incubation of nematodeswith the candidates; “24-24” indicates observation after incubation withcandidates for 24 h and then 24 h in water; “48-24”indicates thatobservation was conducted after incubation with candidates for 48 h andthen 24 h in water; 72-24indicates that observation was conducted afterincubation with candidates for 72 h and then 24 h in water

Example 10 In Vitro Dose-Response Assay Against Free-Living Nematodes

To evaluate the efficacy and stability of B. megaterium H491 whole cellbroth, supernatant was tested for effect on J2 motility at differentdilutions: 1×=full strength; 0.75×=75 ml H491 supernatant: 25 ml water;0.50×=50 ml H491 supernatant: 50 ul water; 0.25×=25 ml H491 supernatant:75 ml water. The motility of juvenile root-knot nematodes (J2's) wasmeasured in an in vitro 96-well plastic cell-culture plate dose-responsebioassay. This procedure is based on a visual grading of motility of thenematodes in each well treated with H491 where each treatment is testedin well repetitions of four: 100 ml of each microbial sample dilutionwas dispensed into its corresponding well followed by 30 ml of a 1:100dilution of plant preservative material (PPM) solution that temporarilysuppresses microbial growth. Finally, 50 ml of nematode solution,containing approximately 15 M. hapla J2 nematodes, was added and theplate was covered and incubated at 25° C. for 24 hours. The effect ofeach dilution sample was then visually scored and recorded. The results,shown in FIG. 6, indicate that the effect on motility ranged from about15% non-motile at a four-fold dilution (0.25×), to 100% non-motile withfull-strength supernatant (1×).

Example 11 Greenhouse Assays

A: Greenhouse Pot Assay: Cucumber with Meloidogyne incognita

To demonstrate the nematicidal activity of H491 supernatant against rootknot nematodes (Meloidogyne incognita), a greenhouse study on cucumber(Cucumis sativus) cv. White Wonderwas conducted using a supernatant asthe test product. One cucumber plant per pot was planted in autoclavedsand and grown in a greenhouse under natural day light. Two-week-oldplants were treated with an 40-mL aliquot of the test product, afterwhich 2000 fresh hatched M. incognita J2s were inoculated into each pot.A week later, a second 40 mL portion of the test product was applied atthe same rate as before. Water, media blank, and Avid (0.1%) were usedas negative, negative, and positive controls, respectively. There werefive replicates for each test product, and the experiment was arrangedin a randomized complete block design. Plants were grown in a greenhousefor four more weeks, after which each plant was harvested and evaluatedfor fresh shoot and root weights. The number of nematodes in each pot,and the number of root gallson each plant, were recorded.

Data presented in Table 5 below show that, although cucumber shoot androot fresh weights from plants treated with H491 supernatant were notstatistically different from untreated controls, the pots treated withH491 supernatant contain significantly fewer nematodes than theuntreated control pots. In addition, root gall number in H491supernatant-treated cucumber plants was significantly less than inplants treated with water or media blank control. These results indicatethat H491 supernatant is effective in restraining gall formation by M.incognita in cucumber.

TABLE 5 Effects H491 supernatant on Meloidogyne incognita Infestation inCucumber in a Greenhouse Pot Assay. Root Weight Nematode Treatment ShootWeight (g) (g) Gall Number Number Avid 15.2 ± 4.4 5.2 ± 2.1    0 ± 0 d316 ± 193 Media 24.3 ± 11.7  4.1 ± 2.5 19.6 ± 3.6 354 ± 195 Water 21.1 ±2.6 6.3 ± 1.2 29.0 ± 8.2 510 ± 256 H491 22.9 ± 5.6 5.6 ± 0.7 10.8 ± 2.8332 ± 161 Data shown are means from two studies.

B: Greenhouse Pot Assay: Effect of H491 Supernatant on MeloidogyneHapla-Infestation of Cucumber and Tomato Plants

Greenhouse studies on cucumber (Cucumis sativus) cv. White Wonder andtomato (Solanum lycopersicum) cv. Roma were performed to demonstrate thenematicidal activity of H491 supernatant on root knot nematodes (M.hapla). One cucumber or tomato plant per pot was planted in autoclavedsand and grown in a greenhouse under natural day light. Pots containingtwo-week-old cucumber or three-week-old tomato plants were treated witha 40-mL aliquot of the test product per pot, after which 2000 fresh M.hapla juveniles were inoculated into each pot. A week later, a secondtreatment of the test product was applied to the pots at the same rateas before. Water, media, and Avid (0.1%) were used as negative,negative, and positive controls, respectively. There were fivereplicates for each test product, and the experiment was arranged in arandomized complete block design. Plants were grown in a greenhouse forfour more weeks for cucumber and six more weeks for tomato, after whicheach plant was harvested and evaluated for fresh shoot and root weightsand shoot height. Plant vigor and root galling index were rated on a0-10 scale. For plant vigor, 0 represents dead and 10 represents mosthealthy; for root gall index, 0 represents no gall, and 10 represents100% of roots galled. The number of nematodes in each pot was alsorecorded.

Results of the first trial are presented in Tables 6 and 7 for cucumberand tomato, respectively. H491 supernatant significantly reduced theroot gall index in cucumber and the number of nematodes in cucumber andtomato compared with the water control. In the second trial, root gallindex and number of nematodes were reduced by H491 in cucumber but nottomato (Tables 8 and 9). The results of both trials indicate that H491supernatant reduces the number of galls in plants, and decreases thedamage in plants from root-knot nematodes.

TABLE 6 Effects of H491 supernatant on Meloidogyne hapla in cucumber ina greenhouse pot assay trial one. Treatment Plant Vigor^(y) Root GallIndex^(z) Nematode Population H491 9 ± 1 2 ± 1 2233 ± 924 Avid (1%) 0 ±0 0 ± 0  0 ± 0 Media 3 ± 1 7 ± 3  5233 ± 1097 Water 1 ± 1 8 ± 1 5467 ±611 Data shown are means from two studies ^(y)Plant vigor on a 0-10scale: 0: dead, 10: the healthiest ^(z)Root gall index on a 0-10 scale:0: no gall, 10: 100% of roots galled

TABLE 7 Effects of H491 supernatant on Meloidogyne hapla in tomato in agreenhouse pot assay trial one. Shoot Shoot Root Root Gall NematodeTreatment Height (cm) Weight (g) Weight (g) Index Number H491  26 ±1^(z) 17 ± 3 4.3 ± 2 2 ± 1 140 Avid (1%)  8 ± 8  1 ± 1 0.4 ± 1 0 ± 0 0Media 31 ± 5 24 ± 4 4.8 ± 3 2 ± 2 267 Water 28 ± 6 14 ± 4 4.2 ± 1 2 ± 1325 Data shown are means from two studies

TABLE 8 Effects H491 supernatant on Meloidogyne hapla in cucumber in agreenhouse pot assay trial two. Nematicidal Root gall Nematode candidateTop weight Root weight index number Avid 16.1 ± 4.5  4.9 ± 2.5 0.2 ± 0.41180 ± 920 Media 8.3 ± 2.6 2.1 ± 1.5 6.0 ± 1.2  3260 ± 1590 Water 5.9 ±1.2 0.5 ± 0.3 7.2 ± 1.3 2580 ± 934 H491 9.3 ± 2.1 1.4 ± 0.7 2.6 ± 1.01500 ± 412 Data shown are means from two studies

TABLE 9 Effects H491 supernatant on Meloidogyne hapla in tomato in agreenhouse pot assay trial two. Nematicidal Root Root gall Nematodecandidate Top weight weight index number Avid 16.9 ± 4.8 6.2 ± 2.1 0 ± 0332 ± 385 Media 21.5 ± 5.9 8.3 ± 1.3 2.2 ± 2.8 342 ± 355 Water 14.1 ±1.1 5.7 ± 1.7 4.1 ± 3.2 334 ± 241 H491 11.3 ± 4.6 4.4 ± 2.3 2.8 ± 3.8332 ± 491 Data shown are means from two studies

Example 12 Immobilization of Nematodes in Infested Soil

In this example, the effect of B. megaterium H491 supernatant on themotility and viability of nematodes in infested soil was determined.These trials were conducted with field soils infested predominantly withsting or lance nematodes. Each trial consisted of 4 treatments: H491supernatant, growth medium blank, a positive control (i.e. 0.1% Avid®)and a negative control (i.e. water). The trials were conducted as arandomized complete block design with 8 replications. Twelve liters ofsoil containing the desired nematodes were collected, separated fromturf and roots, and homogenized. After mixing the soil, 5 (100 cm³) soilsamples were extracted using a centrifugation-sugar flotation methodand, for each sample, the number of nematodes of each type was countedto ensure even distribution of the major nematodes of interest (sting orlance) throughout the soil. Next, 200 cm³ portions ofnematode-containing soil were measured out and placed into a 2×2×2-inchplastic pot. The treatments were applied as a drench treatment (40ml/pot). The pots were then placed on a lab bench and left for 72 hoursto expose the nematodes to the treatments.

Next, the soil from each pot was washed onto a modified Baermannapparatus for nematode extraction. The Baermann apparatus allows livenematodes to move out of the soil, through a filter, and into water,where they can be counted. Dead or immotile nematodes remain in thesoil. After incubating the Baermanns for 96 hours, the live nematodeswere collected and counted. Nematode counts from each treatment werecompared with SAS 9.2 using Fisher's LSD mean separation at P≦0.05 forall nematodes observed.

The effects of H491 supernatant on four plant-parasitic nematode generawere determined. These four genera, observed in the field soil used toconduct the sting bench trial, were: sting nematode, lance nematode,Peltamigratus sp. and Scutellonema sp. Sting nematode counts wereincreased by 5% in soil exposed to growth medium, compared to soilexposed only to water. Compared to negative control (soil exposed towater) soil exposed to Avid or H491 supernatant experienced a 14%decrease in sting nematode number.

Peltamigratus sp. counts were decreased by 7%, 32%, and 88% aftertreatment of soil with medium, H491 supernatant and Avid, respectively;compared to the negative control (water). Scutellonema sp. counts wereunchanged in the soil treated with growth medium, compared to thenegative control (water), and decreased by 17% and 75% in soil treatedwith H491 and Avid, respectively, compared to the negative control(water). Lance nematode counts were decreased by 9%, 35%, and 69% insoil subjected to treatments with medium, H491 supernatant, and Avid,respectively, compared to the water control.

Example 13 Extraction and Isolation of Compounds from B. Megaterium H491Culture Supernatant

The following procedure was used for the purification of compounds fromcultures of B. megaterium. Briefly, a crude extract of B. megateriumH491 culture broth was extracted with Amberlite, and the crude extractwas fractionated by vacuum liquid chromatography. An active VLC fractionwas further fractionated by HPLC, and active compounds were identified.The nematicidally active compound 4-Phenylbutanoic acid (Compound 1) wasobtained from VLC fraction 3, as described below.

Liquid Chromatography of Crude Extract

The culture broth obtained from a 10-L fermentation of B. megaterium inV-8-broth was extracted with Amberlite XAD-7 resin (Asolkar et al.,2006) by shaking the cell suspension with resin at 225 rpm for two hoursat room temperature. The resin and cell mass were collected byfiltration through cheesecloth and washed with deionized water to removesalts. The resin, cell mass, and cheesecloth were then soaked for 2 h inacetone/methanol (50/50) after which the acetone/methanol was filteredand dried under vacuum, using a rotary evaporator, to yield a crudeextract. The crude extract was fractionated by reversed-phase C18 vacuumliquid chromatography (H₂O/CH₃OH; gradient 80:20% to 0:100%) into 6fractions (FIG. 1). These fractions were concentrated to dryness using arotary evaporator, and the resulting dry residues were screened forbiological activity using M. incognita and M. hapla.

Assay for Biological Activity of VLC Fractions of Bacillus megateriumExtract

VLC fractions 1-6 were dissolved in dimethylsulfoxide (DMSO) and testedin an in vitro 96-well plastic cell-culture plate extract bioassay toidentify the fraction/s that contain the desired active metabolite/s. Totest for biological activity, 15-20 J2 nematodes in 50 ml of water wereexposed to 100 ml of a 4 mg/ml fraction concentrate for 24 hours at 25°C. Each fraction was tested in quadruplicate. Results were recordedbased on a visual grading of immotility of the juvenile nematodes (J2's)in each well. Results are shown in FIG. 7.

Reverse Phase HPLC Fractionation of VLC Fractions

The active fractions were subjected to reversed phase HPLC on a SpectraSystem P4000 system (Thermo Scientific) to provide pure compounds, whichwere then screened in bioassays as described above to identify activecompounds. To confirm the identity of the compounds, additionalspectroscopic data such as LC/MS and NMR were recorded.

Fraction 3 (above) was applied to a HPLC C-18 column (Phenomenex, Luna10u C18(2) 100 A, 250×30), and the column was developed with awater:acetonitrile gradient solvent system (0-10 min, 80-70% aqueousCH₃CN; 10-45 min, 70-50% aqueous CH₃CN; 45-55 min, 35-30% aqueous CH₃CN;55-60 min, 30-20% aqueous CH₃CN; 60-65 min, 20-0% aqueous CH₃CN; 65-75min, 100% CH₃CN; 75-80 min, 0-80% aqueous CH₃CN) at 7 mL/min flow ratewith UV detection at 210 nm. The active compound 4-Phenylbutanoic acid(1), had a retention time 47.85 min.

Assay or Biological Activity of HPLC Fractions

Each HPLC peak was tested in an in vitro 96-well plastic cell-cultureplate extract bioassay in order to identify the peak/s that contain thedesired active metabolite/s. For these assays, 15-20 nematodes in a 50ml water solution were exposed to 3 ml of a 20 mg/ml peak concentratefor a 24 hour period at 25° C. Once the incubation period was completed,results were recorded based on a visual grading of immotility of thejuvenile nematodes (J2's) in each well treated with H491 purified peaksH1-H38; each treatment was tested in well repetitions of four. Theresults, shown in FIG. 8, indicate that H491 produces numerous compoundswith pesticidal activity. Fraction 29 was selected for further analysisand testing.

Mass Spectroscopic Analysis of Compounds

Mass spectroscopy of active peaks, isolated by HPLC using the procedureset forth above, was performed on a Thermo Finnigan LCQ Deca XP Pluselectrospray (ESI) instrument using both positive and negativeionization modes in a full scan mode (m/z 100-1500 Da) on a LCQ DECAXP^(plus) Mass Spectrometer (Thermo Electron Corp., San Jose, Calif.).Thermo high performance liquid chromatography (HPLC) instrument equippedwith Finnigan Surveyor PDA plus detector, autosampler plus, MS pump anda 4.6 mm×100 mm Luna C18 5μ 100 A column (Phenomenex). The solventsystem consisted of water (solvent A) and acetonitrile (solvent B). Themobile phase begins at 10% solvent B and was linearly increased to 100%solvent B over 20 min and then kept for 4 min, and finally returned to10% solvent B over 3 min and kept for 3 min. The flow rate was 0.5mL/min. The injection volume is 10 μL and the samples were kept at roomtemperature in an auto sampler. The compounds were analyzed by LC-MSutilizing the LC and reversed phase chromatography. Mass spectroscopyanalysis of the present compounds is performed under the followingconditions: The flow rate of the nitrogen gas was fixed at 30 and 15 arbfor the sheath and aux/sweep gas flow rate, respectively. Electrosprayionization was performed with a spray voltage set at 5000 V and acapillary voltage at 35.0 V. The capillary temperature was set at 400°C. The data was analyzed on Xcalibur software. The 4-Phenylbutanoic acid(1) exhibited a molecular mass of 163.34 in negative ionization mode(see FIG. 2).

NMR Spectroscopic Analysis of Compounds

NMR spectra were measured on a Bruker 600 MHz gradient fieldspectrometer. The reference is set on the internal standardtetramethylsilane (TMS, 0.00 ppm).

For structure elucidation, the purified 4-Phenylbutanoic acid (1) withmolecular weight 164 was further analyzed using a 600 MHz NMRinstrument. ¹H NMR δ values at 7.28 (2H), 7.19 (2H), 7.17, 2.67, 2.31,and 1.92 were detected (see FIG. 3). ¹³C NMR values of 177.5, 142.9,129.5, 129.5, 129.4, 129.4, 126.9, 36.1, 34.4, and 28 were detected (seeFIG. 4). The active compound was isolated as a colorless solid, with UVabsorption bands at 220 & 283 nm. The negative electrospray ionizationmass spectrum revealed molecular ion peak at m/z 163.32 [M-H]⁻ and327.14 [2M-H]⁻ suggesting a molecular weight of 164, which is consistentwith the molecular formula C₁₀H₁₀O₂ (with 5 degrees of unsaturation).The ¹H NMR spectrum exhibited signals indicative of the presence of amono substituted aromatic ring (δ 7.28, 2H, d, J=7.7 Hz; 7.19, 2H, d,J=7.7 Hz; 7.17 1H, t, J=7.7 Hz). Furthermore, the ¹H NMR spectrumrevealed the presence of three methylene groups, at δ 2.62, 2H, t,J=7.92 Hz, 2.30, 2H, t, J=7.65 and 1.92, 2H, q, J=7.65 Hz. The ¹³C NMRspectrum of Compound (I), interpreted with the help of the HSQCexperiment, indicated the presence of 10 carbon atoms, including onecarbonyl (δ_(c) 177.5); six sp² carbons, one of which is unprotonated(δ_(c) 142.9); five protonated carbon atoms (δ_(c) 129.5, 129.5, 129.4,129.4, 126.9); and three methylenes (δ_(c) 36.1, 34.4, 28.1). Theanalysis of the COSY and HSQC correlation revealed two spin systems, oneinvolving three methylene groups of the structure —CH₂—CH₂—CH₂—, and theother comprising a mono substituted benzene ring. From the detailedanalysis of COSY, HMQC and HMBC experiments, the structure for thecompound was assigned as 4-phenylbutanoic acid. The assigned structurewas confirmed by comparison with a synthetic sample. This compound hasbeen isolated from the marine bacterium B. pumilus S6-15, which inhibitsbiofilm formation in gram positive and gram negative species (Nithya etal., 2011).

Example 14 Greenhouse Cone Assay: Effect of Meloidogyne incognita onTomato Seedlings

Greenhouse studies on tomato (Solanum lycopersicum) cv. UC 82 wereperformed to test the nematicidal activity of H491 supernatant on rootknot nematodes (M. incognita). One 25-day-old tomato seedling per conecontainer (Stuewe & Sons, Inc, Tangent, Oreg.) was transplanted intoautoclaved sand and soil mix (2:1) and grown in a greenhouse undernatural day light. 16 days after transplanting, each tomato seedlings(3-4 true leaves) was drenched with a 10-ml aliquot of whole cell broth(WCB) of H491 and other test products by pouring the aliquot directlyonto the soil surface. Thereafter, 4000 eggs of M. incognita suspendedin 1 ml of water were inoculated into three 6-cm-deep holes around thestems in each cone. A week later, a second drench was applied to thecones at the same rate as the first drench. Water and Avid (625 ppm)were used as negative and positive controls, respectively. There werefour replicates and the experiment was arranged in a randomized completeblock design. Plants were grown in a greenhouse for 3 weeks before beingtaken down. Each plant was then evaluated for phytotoxicity, fresh top(or shoot) weight, fresh root weight, gall numbers per root and gallindex based on a 0 to 10 scale (0=no galls, 10=100% of roots galled).There was no phytotoxicity observed during the incubation period of thetest. Statistical analysis (ANOVA) was performed using Fisher test, andthe statistical differences among treatment means were calculated atP≦0.05.

Results are presented in Table 10. H491 WCB significantly reduced thegall index, number of galls per root and number of galls per gram ofroot compared with the water control. The plants treated with H491 hadheavier fresh topweight than those treated with water. No significantdifference was observed on the fresh root weight between treatments.

TABLE 10 Effects of H491 whole cell broth (WCB) on Meloidogyne incognitain tomato in a greenhouse cone assay^(x) galls/g of Treatment FTW(g)^(y) FRW (g)^(y) gall index^(z) galls/root root H491 10.74 ± 0.34^(a) 6.1 ± 0.59^(a) 2.3 ± 0.96^(b) 142.5 ± 69.70^(b) 22.7 ± 9.28^(b) water 7.35 ± 6.03^(b) 5.43 ± 1.37^(a) 6.5 ± 1.29^(a) 271.8 ± 53.60^(a) 54.1 ±25.20^(a) avid  8.43 ± 1.36^(ab) 5.33 ± 1.33^(a)   0 ± 0.00^(c)  1.0 ±2.00^(c) 0.24 ± 0.48^(b) p-value 0.038 0.606 0 0 0.003 ^(x)Data aremeans ± standard deviations (SD) with 4 replicates. The values followedby the same letter in the same column were not significantly different,according to Fisher's least significant difference at P ≦ 0.05. ^(y)FTW:fresh top weight; FRW: fresh root weight. ^(z)gall index on a 0 to10scale: 0: no gall; 10: 100% of roots galled.

Example 15 Dose Response for H491 Whole Cell Broth in Mini Tube inPlanta Bioassay

H491 Bacillus megaterium whole cell broth (WCB) was tested in an inplanta bioassay at 3 different dilutions in water (1×, 0.7×, and 0.5×v/v) to determine the potency of the WCB against Meloidogyne incognitajuveniles. This test is a miniature greenhouse pot test in which sterilefalcon tubes were modified by cutting the top 1.5 inches off and makingholes in the bottom to allow drainage. The tubes were filled with amixture of sand and soil (1:2) and drenched with 2 mL aliquots of H491whole cell broth (50, 70 and 100%), Avid (label rate) as positivecontrol, and water as negative control. After drenching, cucumber seeds(cv. SMR58) were planted and then the tubes were inoculated with 800eggs of M. incognita. The tubes were covered with parafilm and placed ina box with wet paper towels to ensure high humidity.

After 4 days, most of the seed had germinated and the parafilm cover wasremoved. After one week, a second dose (1 mL) was applied. The test wasterminated after two weeks and fresh top weight, fresh root weight andgall index (scale: 1 unhealthy roots-10 healthy roots) were assessed.

The results, shown in FIG. 10, indicate that gall index was reduced atall concentrations of H491 whole cell broth tested.

Example 16 In Vitro Motility Assay

H491 WCB obtained from four separate fermentation batches was tested inan in vitro motility assay. In this assay, M. incognita juveniles (J2)were exposed to test samples in a 96-well flat bottom plate. After 24hours the juveniles were transferred into 6-well plates containing 1.5%water agar, to allow potential recovery. Motility was measured bycounting the number of juveniles (J2) that moved out of the initial areaafter another 24 hours. The results are shown in FIG. 11. The dataindicates that, for all four batches of H491 WCB, 100% immobilizationwas achieved after 24 hour exposure.

Example 17 Plant Growth Promotion Capabilities of B. Megaterium H491

B. megaterium H491 was tested for plant growth promotion markers on fivedifferent plate assays, and representative results are shown in Table11. The bacterium was able to solubilize phosphate and produced theenzyme ACC deaminase. It was also capable of growing on methanol ascarbon source. These results suggested that B. megaterium H491 canpromote growth and confer stress tolerance to plants.

TABLE 11 Phosphate sol. ACC deaminase IAA* CAS* AMS* H491 +++ +++ + − ++*IAA: indole acetic acid; CAS: Chrome azurol S; AMS: Ammonium mineralsalts

Example 18 Growth Promoting Activity on Soy and Sorghum

To test for the ability of B. megaterium H491 to promote plant growth,two seedling vigor assays were performed on soy and sorghum. Strain H491was grown on an agar plate and a few colonies were transferred to 50 mlsterile Luria broth (LB: 25 g/l) and incubated at 25° C., 180 rpm for 24h. Bacterial cells were harvested from the LB cultures by centrifugationat 3220×g for 20 minutes. The supernatant was discarded and the cellswere washed in 20 ml sterile MgSO₄ buffer then centrifuged for a secondtime at 3220×g for 20 minutes. After discarding the supernatant, thecells were re-suspended in a small volume of sterile buffer. Theconcentration of cells in the suspension was determined by measuring theabsorbance at 600 nm in a spectrophotometer. Seeds were treated with thecell suspension by imbibition. A cell inoculum (1×10⁸ CFU/ml) wasdispensed over the seeds, in a 50 ml Falcon tube, at a rate of 0.6 mlper gram of seed (for seed with 250-300 seed/g). The seed and inoculumwere incubated overnight at 25° C., and treated seeds were then dried ina sterile hood for 30 min. The negative control was prepared in the samemanner, except the cell inoculum was replaced with sterile buffer.Growth promotion was evaluated by measuring fresh weight of seedlings,as shown in Table 12. For soy, the seedling weight doubled with the H491treatment. For sorghum, a 64% increase of the fresh weight was observed.

TABLE 12 Control (Buffer) H491 treated Soy 5.46 11.22 Sorghum 0.89 1.46

Example 19 Growth Promoting Activity on Corn

Corn seeds were planted and drenched at planting time and one week afterplanting with B. megaterium H491 whole cell broth (WCB). A total of 10plants per pot and 9 pots per treatment were planted and evaluated.Total fresh weight was recorded 2 weeks after planting, and statisticalanalysis was performed using Minitab ANOVA Tukey's.

The results are shown in Table 13. In corn plants treated with H491 WCB,a significant increase in vegetative fresh weight was observed, with a95% simultaneous confidence interval as assessed by Tukey's.

TABLE 13 Whole cell broth Corn Control H491 AVG 21.48 30.07 SD 4.02 2.42

Example 20 Effect of Compound 1 on Nematode Motility

HPLC fraction H29 (Example 13 and FIG. 8) was determined to be4-phenylbutanoic acid. In this example, 4-phenylbutanoic acid(Compound 1) was synthesized and tested for dose-response in a nematodemotility assay. M. incognita juveniles (J2) were exposed to differentconcentrations of 4-phenylbutanoic acid in a 96-well flat bottom platefor 24 hours. Juveniles were then transferred into 6-well platescontaining 1.5% water agar to allow recovery. After a further 24 hours,motility was determined by counting the number of juveniles (J2) thathad moved out of the initial area. Water and DMSO were used as negativecontrols, and Avid as a positive control.

The results are shown in FIG. 12. Using 75% immotility as a thresholdfor nematicidal activity, concentrations of 4-phenylbutanoic acid of0.017 mg/ml or greater provided substantial nematicidal activity.

Example 22 Nematicidal Activity of Three Isolates of B. Megaterium

Isolates of B. megaterium H491, M018 and J142 were cultured. Whole cellbroth from each of the three cultures was tested in the water agarimmotility assay described in Example 21. The results, shown in FIG. 13,indicate that whole cell broth from all three of the strains are able torender essentially 100% of juveniles immotile.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Culture Collection(NRRL), 1815 N. University Street, Peoria, Ill. 61604 USA, and given thefollowing number:

Deposit Accession Number Date of Deposit Bacillus megaterium strain H491NRRL B-50769 Aug. 3^(rd), 2012 Bacillus megaterium strain M018 NRRLB-50770 Aug. 3^(rd), 2012 Bacillus megaterium strain J142 NRRL B-50769Aug. 3^(rd), 2012

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentaction.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

REFERENCES

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What is claimed is:
 1. An isolated strain of a Bacillus megaterium whichhas the following characteristics: (A) a 16S rRNA gene sequencecomprising at least one of: (1) a forward sequence having at least 99%identity to the sequence set forth in SEQ ID NO:1, a reverse sequencehaving at least 99% identity to the sequence set forth in SEQ ID NO:2and at least 99% identity to the consensus sequence set forth in SEQ IDNO: 3; (2) a forward sequence having at least 99% identity to thesequence set forth in SEQ ID NO:4, a reverse sequence having at least99% identity to the sequence set forth in SEQ ID NO:5 and at least 99%identity to the consensus sequence set forth in SEQ ID NO: 6; (3) aforward sequence having at least 99% identity to the sequence set forthin SEQ ID NO:7, a reverse sequence having at least 99% identity to thesequence set forth in SEQ ID NO:8 and at least 99% identity to theconsensus sequence set forth in SEQ ID NO: 9; (B) a recA gene sequencecomprising at least one of: (1) a forward sequence having at least 99%identity to the sequence set forth in SEQ ID NO:14, a reverse sequencehaving at least 99% identity to the sequence set forth in SEQ ID NO:15and at least 99% identity to the consensus sequence set forth in SEQ IDNO: 16; (2) a forward sequence having at least 99% identity to thesequence set forth in SEQ ID NO:17, a reverse sequence having at least99% identity to the sequence set forth in SEQ ID NO:18 and at least 99%identity to the consensus sequence set forth in SEQ ID NO: 19; (3) aforward sequence having at least 99% identity to the sequence set forthin SEQ ID NO:20, a reverse sequence having at least 99% identity to thesequence set forth in SEQ ID NO:21 and at least 99% identity to theconsensus sequence set forth in SEQ ID NO: 22; (C) pesticidal activity;(D) production of a pesticidal compound having the following properties:(1) a molecular weight of about 140-185 as determined by LiquidChromatography/Mass Spectroscopy (LC/MS); (2) ¹H NMR values of δ 7.28(2H), 7.19 (2H), 7.17, 2.67, 2.31, 1.92 and has ¹³C NMR values of 177.5,142.9, 129.5, 129.5, 129.4, 129.4, 126.9, 36.1, 34.4, 28.1; (3) a HighPressure Liquid Chromatography (HPLC) retention time of about 8-18minutes, on a reversed phase C-18 HPLC (Phenomenex, Luna 5μ C18(2) 100A, 100×4.60 mm) column using a water:acetonitrile (CH₃CN) with agradient solvent system (0-20 min; 90-0% aqueous CH₃CN, 20-24 min; 100%CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90% aqueous CH₃CN) at0.5 mL/min flow rate and UV detection at 210 nm; and (E)non-pathogenicity to vertebrate animals.
 2. The strain of claim 1,wherein the strain is susceptible to one or more of an antibioticselected from the group consisting of tetracycline, erythromycin,streptomycin, penicillin, ampicillin, Oxytetracycline, Chloramphenicol,Ciprofloxacin, Gentamicin, Piperacillin, Imipenem andSulphamethoxazole-Trimethoprim.
 3. A supernatant, filtrate, extract,compound, metabolite, or cell fraction obtained from the bacterium ofclaim
 1. 4. The strain of claim 1, wherein the Bacillus megateriumstrain has the identifying characteristics of B. megaterium strain H491(NRRL Accession No. B-50769), Bacillus megaterium strain M018 (NRRLAccession No. B-50770) and Bacillus megaterium strain J142 (NRRLAccession No. B-50771).
 5. A substantially pure culture or whole cellcomprising the strain of claim 1 or an extract, cell fraction, compound,supernatant, metabolite or filtrate derived therefrom.
 6. A combinationcomprising (a) a first substance, wherein said first substance is asubstantially pure culture or whole cell comprising the strain of claim1 or an extract, cell fraction, compound, supernatant, metabolite orfiltrate derived therefrom; (b) optionally a second substance, whereinsaid second substance is a chemical or biological pesticide and (c)optionally at least one of a carrier, diluent, surfactant, or adjuvant.7. The combination according to claim 6, wherein said second substanceis a pesticide selected from the group consisting of a nematicide, afungicide and an insecticide.
 8. The combination according to claim 6wherein the combination is a composition.
 9. A method for modulatingpest infestation in a plant, the method comprising applying to the plantand/or seeds thereof and/or substrate used for growing said plant anamount of the combination of claim 6 effective to modulate said pestinfestation.
 10. A method for isolating a compound which (1) has amolecular weight of about 140-185 as determined by LiquidChromatography/Mass Spectroscopy (LC/MS); (2) has ¹H NMR values of δ7.28 (2H), 7.19 (2H), 7.17, 2.67, 2.31, 1.92 and has ¹³C NMR values of177.5, 142.9, 129.5, 129.5, 129.4, 129.4, 126.9, 36.1, 34.4, 28.1; (3)has an High Pressure Liquid Chromatography (HPLC) retention time ofabout 8-18 minutes, on a reversed phase C-18 HPLC (Phenomenex, Luna 5μC18(2) 100 A, 100×4.60 mm) column using a water:acetonitrile (CH₃CN)with a gradient solvent system (0-20 min; 90-0% aqueous CH₃CN, 20-24min; 100% CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90% aqueousCH₃CN) at 0.5 mL/min flow rate and UV detection of 210 nm.; and (4) haspesticidal properties, the method comprising: (a) culturing the Bacillusmegaterium strain of claim 1 for a time and at a temperature sufficientto produce said compound; and (b) isolating said compound produced in(a) from said culture.
 11. A combination comprising: (A) a compoundwhich (1) has a molecular weight of about 140-185 as determined byLiquid Chromatography/Mass Spectroscopy (LC/MS); (2) has ¹H NMR valuesof δ 7.28 (2H), 7.19 (2H), 7.17, 2.67, 2.31, 1.92 and has ¹³C NMR valuesof 177.5, 142.9, 129.5, 129.5, 129.4, 129.4, 126.9, 36.1, 34.4, 28.1;(3) has a High Pressure Liquid Chromatography (HPLC) retention time ofabout 8-18 minutes, on a reversed phase C-18 HPLC (Phenomenex, Luna 5μC18(2) 100 A, 100×4.60 mm) column using a water:acetonitrile (CH₃CN)with a gradient solvent system (0-20 min; 90-0% aqueous CH₃CN, 20-24min; 100% CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90% aqueousCH₃CN) at 0.5 mL/min flow rate and UV detection of 210 nm.; and (4) haspesticidal properties; (B) at least one of (1) a second substance,wherein said second substance is a chemical or biological pesticide or(2) at least one of a carrier, diluent, surfactant, or adjuvant.
 12. Thecombination according to claim 11, wherein the combination is acomposition.
 13. A method for modulating pest infestation in a plantcomprising applying to the plant and/or seeds thereof and/or substrateused for growing said plant an amount of (a) a compound which (1) has amolecular weight of about 140-185 as determined by LiquidChromatography/Mass Spectroscopy (LC/MS); (2) has ¹H NMR values of δ7.28 (2H), 7.19 (2H), 7.17, 2.67, 2.31, 1.92 and has ¹³C NMR values of177.5, 142.9, 129.5, 129.5, 129.4, 129.4, 126.9, 36.1, 34.4, 28.1; (3)has a High Pressure Liquid Chromatography (HPLC) retention time of about8-18 minutes, on a reversed phase C-18 HPLC (Phenomenex, Luna 5μ C18(2)100 A, 100×4.60 mm) column using a water:acetonitrile (CH₃CN) with agradient solvent system (0-20 min; 90-0% aqueous CH₃CN, 20-24 min; 100%CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90% aqueous CH₃CN) at0.5 mL/min flow rate and UV detection of 210 nm.; and (4) has pesticidalproperties; and (b) optionally another substance, wherein said substanceis a pesticide, effective to modulate said pest infestation and/oremergence or growth of monocotyledonous, sedge or dicotyledonous weeds.14. The method according to claim 13, wherein said compound (I) has thestructure selected from the group consisting of: (B) ##STR001##

or a pesticidally acceptable salt or stereoisomers thereof, wherein n is0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; X is O, NH, NR or S; R is lower chainalkyl containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl moieties, aryl orarylalkyl moiety, substituted lower alkyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl,substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl,substituted thioalkyl, hydroxy, halogen, amino, amido, or carboxyl; R₁,R₂, R₃, R₄, R₅, R₆ are each independently H, hydroxyl, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, cycloalkyl, substitutedcycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substitutedthioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl,oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; (C) ##STR001a##

or a pesticidally acceptable salt or stereoisomers thereof, wherein X isO, NH, NR or S; R is lower chain alkyl containing 1, 2, 3, 4, 5, 6, 7, 8or 9 alkyl moieties, aryl or arylalkyl moiety, substituted lower alkyl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, or carboxyl; n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R₁, R₂, R₃, R₄are each independently H, hydroxyl, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl; and (D)##STR001b##

or a pesticidally acceptable salt or stereoisomers thereof, wherein n is0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R₁, R₂, R₃, R₄, R₅ are eachindependently H, hydroxyl, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl.
 15. The method according to claim 13, whereinsaid compound (I) is 4-phenylbutanoic acid.
 16. A seed comprising thestrain of claim 1, the combination of claim 11 or a compound which: (1)has a molecular weight of about 140-185 as determined by LiquidChromatography/Mass Spectroscopy (LC/MS); (2) has ¹H NMR values of δ7.28 (2H), 7.19 (2H), 7.17, 2.67, 2.31, 1.92 and has ¹³C NMR values of177.5, 142.9, 129.5, 129.5, 129.4, 129.4, 126.9, 36.1, 34.4, 28.1; (3)has an High Pressure Liquid Chromatography (HPLC) retention time ofabout 8-18 minutes, on a reversed phase C-18 HPLC (Phenomenex, Luna 5μC18(2) 100 A, 100×4.60 mm) column using a water:acetonitrile (CH₃CN)with a gradient solvent system (0-20 min; 90-0% aqueous CH₃CN, 20-24min; 100% CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90% aqueousCH₃CN) at 0.5 mL/min flow rate and UV detection of 210 nm.; and (4) haspesticidal properties.
 17. A method for modulating (i) growth of aplant, and/or (ii) germination of a seed of said plant, the methodcomprising contacting said plant and/or its seed and/or its growthsubstrate with a composition selected from the group consisting of: (a)the bacterium of claim 1; (b) a supernatant, filtrate, extract,compound, metabolite, or cell fraction obtained from the bacterium ofclaim 1; and (c) a compound which (1) has a molecular weight of about140-185 as determined by Liquid Chromatography/Mass Spectroscopy(LC/MS); (2) has ¹H NMR values of δ 7.28 (2H), 7.19 (2H), 7.17, 2.67,2.31, 1.92 and has ¹³C NMR values of 177.5, 142.9, 129.5, 129.5, 129.4,129.4, 126.9, 36.1, 34.4, 28.1; (3) has a High Pressure LiquidChromatography (HPLC) retention time of about 8-18 minutes, on areversed phase C-18 HPLC (Phenomenex, Luna 5μ C18(2) 100 A, 100×4.60 mm)column using a water:acetonitrile (CH₃CN) with a gradient solvent system(0-20 min; 90-0% aqueous CH₃CN, 20-24 min; 100% CH₃CN, 24-27 min; 0-90%aqueous CH₃CN, 27-30 min; 90% aqueous CH₃CN) at 0.5 mL/min flow rate andUV detection at 210 nm an amount of said composition effective tomodulate said growth of said plant and/or germination of said seed. 18.The method according to claim 17, wherein the compound has a structureselected from the group consisting of: (a) ##STR001##

or a pesticidally acceptable salt or stereoisomers thereof, wherein n is0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; X is O, NH, NR or S; R is lower chainalkyl containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl moieties, aryl orarylalkyl moiety, substituted lower alkyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl,substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl,substituted thioalkyl, hydroxy, halogen, amino, amido, or carboxyl; R₁,R₂, R₃, R₄, R₅, R₆ are each independently H, hydroxyl, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,heterocyclic, substituted heterocyclic, cycloalkyl, substitutedcycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substitutedthioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl,oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; (b) ##STR001a##

or a pesticidally acceptable salt or stereoisomers thereof, wherein X isO, NH, NR or S; R is lower chain alkyl containing 1, 2, 3, 4, 5, 6, 7, 8or 9 alkyl moieties, aryl or arylalkyl moiety, substituted lower alkyl,heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, or carboxyl; n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R₁, R₂, R₃, R₄are each independently H, hydroxyl, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl; and (c) ##STR001b##

or a pesticidally acceptable salt or stereoisomers thereof, wherein n is0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R₁, R₂, R₃, R₄, R₅ are eachindependently H, hydroxyl, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substitutedalkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino,amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl,sulfonamide, or sulfuryl.
 19. The method according to claim 18, whereinsaid compound is 4-phenylbutanoic acid.
 20. The method according toclaim 17, wherein said other substance is a nematicide.
 21. The methodaccording to claim 17, wherein said plant is selected from the groupconsisting of strawberry, squash, cucumber, tomato, rose, peppereggplant, grapevine, cotton, onion, garlic, wheat, soy, corn and rice.22. The method according to claim 17, further comprising transplantingsaid plant into said growth substrate.
 23. The method according to claim22, wherein prior to transplanting said plant into said growthsubstrate, one or more roots of said plant are treated with saidcomposition.